Modulation of tumor immunity by protein-mediated 02 delivery

ABSTRACT

The invention provides methods to modulate hypoxia-mediated tumor immunity by administration of an O2 carrier polypeptide (e.g., an H-NOX protein). The methods of the invention target both hypoxia inducible factor 1 alpha (HIF-1α) pathways and non-HIF-1α pathways of tumor immunity. Such methods are useful in the treatment of a wide variety of cancers and may be used alone or in combination with other anti-cancer therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/134,523, filed Mar. 17, 2015, the disclosure of whichis hereby incorporated by reference in its entirety for all purposes.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 627042000940SeqList.txt,date recorded: Mar. 17, 2016, size: 41 KB).

TECHNICAL FIELD

This application pertains to the modulation of tumor immunity bydelivering oxygen to the tumor by way of a protein O₂ carrierpolypeptide; for example an H-NOX protein.

BACKGROUND OF THE INVENTION

The hypoxic tumor microenvironment suppresses the host's immuneanti-tumor defenses by modulating multiple signaling pathways including,but not limited to, hypoxia inducible factor (HIF-1) signaling (Codo etal., 2014 Oncotarget, 5(17), 7651-7662; Lee, Mace, & Repasky, 2010 Int JHyperthermia, 26(3), 232-246; Wei et al., 2011 PLoS One, 6(1), e16195).Major hypoxia immunomodulating pathways are summarized in FIG. 1.Briefly, HIF-1 has been shown to: a) activate adenosinergic A2 and PD-L1pathways that inhibit recruitment and activation of helper and killerT-cells and NK cells, key effectors of anti-tumor responses (Noman etal., 2014 J Exp Med, 211(5), 781-790; Ohta et al., 2006 Proc Natl AcadSci USA, 103(35), 13132-13137); b) recruit and activate inhibitoryregulatory T cells (Treg), tumor associated macrophages (TAM) and othermyeloid-derived suppressor cells (MDSC) (Chaturvedi et al., 2014 ProcNatl Acad Sci USA, 111(20), E2120-2129; Corzo et al., 2010 J Exp Med,207(11), 2439-2453; Wei et al., 2011); c) directly inhibit the abilityof tumor cells to be recognized by immune system (Siemens et al., 2008Cancer Res, 68(12), 4746-4753). In addition, HIF-1-dependent and-independent epigenetic mechanisms contribute to inhibition ofanti-tumor immune-responses and enhance tumor growth, angiogenesis andmetastasis (Codo et al., 2014; Mimura et al., 2011 J Pharmacol Sci,115(4), 453-458).

In mouse metastatic tumor models, continuous supplemental oxygenationhas been shown to inhibit tumor growth and prevent tumor's immune escapethrough inhibition of A2AR (A2A adenosine receptor) adenosinergicpathway leading to T and NK cell activation (Hatfield et al., 2015 SciTransl Med, 7(277), 277ra230). Specifically, continuous treatment with60% respiratory oxygen of mice bearing MCA205, B16 or 4T1 pulmonarymetastases resulted in >2-fold decrease in number of metastatic foci andenhanced survival. These data correlated with decrease in tumor andlymphocyte hypoxia, increased activated CD8 T cell (CD8+CD69+CD44+)tumor infiltration, upregulation of immunostimulating cytokines andchemokines and were dependent on intact A2AR signaling. At the same timerespiratory hyperoxia was shown to reduce the number and suppressive theactivity of Treg in pulmonary tumor microenvironment (TME) due toreduced Foxp3, CD39/CD73 (adenosine generating enzymes upstream of A2AR)and CTLA-4 expression. Finally, tumor regression induced by dualCTLA-4/PD-1 blockade of pulmonary tumors was enhanced by continuousrespiratory hyperoxia.

Despite convincing pre-clinical evidence demonstrating the capacity oftumor oxygenation to reverse immunosuppressive TME and inhibit tumorgrowth, in human clinical trials supplemental oxygenation usinghyperbaric or normobaric oxygen yielded limited effects (Overgaard, 2007J Clin Oncol, 25(26), 4066-4074). This is likely due to the inability ofsoluble oxygen to effectively diffuse beyond ˜80 μm from blood vessels,limiting its penetration deep into hypoxic tumor tissue. Therefore, theneed exists for oxygen delivery agents that penetrate into patients'tumors to transport oxygen beyond the normal diffusion limits, andthereby oxygenate hypoxic microenvironments to impede immunosuppressivepathways. This will result in maximal stimulation of anti-tumor immuneresponses, both alone and in combination with other immune checkpointinhibitors and other cancer immunotherapy approaches.

H-NOX proteins (named for Heme-Nitric oxide and OXygen binding domain)are members of a highly-conserved, well-characterized family ofhemoproteins (Iyer, L M et al. (2003) BMC Genomics 4(1):5; Karow, D S etal. (2004) Biochemistry 43(31):10203-10211; Boon, E M et al. (2005)Nature Chem. Biol. 1:53-59; Boon, E M et al. (2005) Curr. Opin. Chem.Biol. 9(5):441-446; Boon, E M et al. (2005) J. Inorg. Biochem.99(4):892-902; Cary, S P et al. (2005) Proc Natl Acad Sci USA102(37):13064-9; Karow D S et al. (2005) Biochemistry 44(49):16266-74;Cary, S P et al. (2006) Trends Biochem Sci 31(4):231-9; Boon, E M et al.(2006) J Biol Chem 281(31):21892-902; Winger, J A et al. (2007) J BiolChem. 282(2):897-907). H-NOX proteins are nitric-oxide-neutral, unlikeprevious hemoglobin-based oxygen carriers, H-NOX do not scavengecirculating nitric oxide (NO), and thus are not associated withhypertensive or renal side effects. The intrinsic low NO reactivity (andhigh NO stability) makes wild-type and mutant H-NOX proteins desirableblood substitutes because of the lower probability of inactivation ofH-NOX proteins by endogenous NO and the lower probability of scavengingof endogenous NO by H-NOX proteins. Importantly, the presence of adistal pocket tyrosine in some H-NOX proteins (Pellicena, P. et al.(2004) Proc Natl. Acad Sci USA 101(35):12854-12859) is suggestive ofundesirable, high NO reactivity, contraindicating use as a bloodsubstitute. For example, by analogy, a Mycobacterium tuberculosishemoglobin protein, with a structurally analogous distal pockettyrosine, reacts extremely rapidly with NO, and is used by theMycobacterium to effectively scavenge and avoid defensive NO produced byan infected host (Ouellet, H. et al. (2002) Proc. Natl. Acad. Sci. USA99(9):5902-5907). However, it was surprisingly discovered that H-NOXproteins actually have a much lower NO reactivity than that ofhemoglobin making their use as blood substitutes possible.

H-NOX proteins for the delivery of O₂ and/or NO for therapeutic andother uses are described in U.S. Pat. Nos. 8,404,631 and 8,404,632; WO2007/139791, WO 2007/139767 and WO 2014/107171; and U.S. patentapplication Ser. No. 14/530,569, the contents of each is incorporated byreference in its entirety.

All references cited herein, including patent applications andpublications, are incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for modulating tumor immunity in anindividual with a tumor comprising administering to the individual aneffective amount of an O₂ carrier polypeptide. In some embodiments, theinvention provides methods for enhancing an immune response to thetumor. In some embodiments, the invention provides methods forincreasing lymphocyte infiltration to a tumor in an individualcomprising administering to the individual an effective amount of an O₂carrier polypeptide. In some embodiments, the increase in lymphocyteinfiltration to the tumor comprises an increase in infiltration of oneor more of CD4 cells, CD8 cells, or NK cells. In some embodiments, theincrease in lymphocyte infiltration to the tumor is accompanied byinhibition of one or more of Treg cells, tumor associated macrophages ormyeloid derived suppressor cells in the tumor. In some embodiments, theincrease in lymphocyte infiltration to the tumor is accompanied by anincrease in MHC1 expression on the tumor cells. In some embodiments, themodulating of tumor immunity comprises increasing antigen processing. Insome embodiments, the modulating of tumor immunity comprises increasingthe presentation capabilities of dendritic cells (DC).

In some embodiments, the invention provides methods for decreasingexpression of hypoxia inducible factor 1α (HIF-1α) and/or hypoxiainducible factor 2α (HIF-2α) in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide. In some embodiments, the invention provides methods fordecreasing expression of programmed death ligand-1 (PD-L1) in a tumor inan individual comprising administering to the individual an effectiveamount of an O₂ carrier polypeptide. In some embodiments, the inventionprovides methods for decreasing expression of A2A adenosine receptor(A2AR) in a tumor in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide.

In some embodiments of the above embodiments, the tumor is a braintumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, atumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, anovarian tumor, a colorectal tumor, a liver tumor, a hepatocellularcarcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, acervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin'slymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, anadrenal tumor, a bladder tumor, a kidney tumor, breast tumor, a multiplemyeloma tumor, a sarcoma, or a squamous cell tumor.

In some aspects, the invention provides methods for treating cancer inan individual comprising administering to the individual an effectiveamount of an O₂ carrier polypeptide. In some embodiments, the cancer isbrain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, melanoma, lung cancer, uterine cancer,ovarian cancer, colorectal cancer, anal cancer, liver cancer,hepatocellular carcinoma, stomach cancer, testicular cancer, endometrialcancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma,esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer,bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma,anal cancer or squamous cell cancer.

In some embodiments of the above aspects and embodiments, the individualis a mammal. In further embodiments, the mammal is a human (e.g., ahuman patient). In other embodiments, the mammal is a pet, a laboratoryresearch animal, or a farm animal. In some embodiments, the pet,research animal or farm animal is a dog, a cat, a horse, a monkey, arabbit, a rat, a mouse, a guinea pig, a hamster, a pig, or a cow.

In some embodiments of the above aspects and embodiments, the O₂ carrierpolypeptide is administered by intravenous, intra-arterial,intratumoral, intravesicular, inhalation, intraperitoneal,intrapulmonary, intramuscular, subcutaneous, intra-tracheal,transmucosal, intraocular, intrathecal, or transdermal administration.In some embodiments, administration of the O₂ carrier polypeptide isrepeated. In some embodiments, administration of the O₂ carrierpolypeptide is repeated daily or twice a day from about 4 weeks to about8 weeks. In some embodiments, the O₂ carrier polypeptide is administeredevery four, every 8, every 12 or every 24 hours for a period of aboutone to about 10 days. In some embodiments, the O₂ carrier polypeptide isadministered as a bolus. In other embodiments, the O₂ carrierpolypeptide is administered by infusion. In some embodiments, the O₂carrier polypeptide is infused in the individual for about 15 minutes,about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6hours, about 12 hours, about 24 hours or more than 24 hours.

In some embodiments, the invention provides methods to modulate tumorimmunity or to treat cancer in an individual wherein an O₂ carrierpolypeptide is administered in combination with radiation therapy. Insome embodiments, the radiation therapy is administered to theindividual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or 24 hours afterthe O₂ carrier polypeptide is administered. In some embodiments, theradiation is X-radiation. In some embodiments, the X-radiation isadministered at about 0.5 gray to about 75 gray. In some embodiments,the administration of the O₂ carrier polypeptide and/or theadministration of the radiation is repeated. In some embodiments, theadministration is repeated more than about any of two, three, fourtimes, five times, ten times, 15 times, 20 times, 25 times or 30 times.In some embodiments, the administration is repeated after one week, twoweeks, three weeks, or four weeks.

In some embodiments, the invention provides methods to modulate tumorimmunity or to treat cancer in an individual wherein an O₂ carrierpolypeptide is administered in combination with chemotherapy orimmunotherapy. In some embodiments, the chemotherapy comprises acytotoxin. In some embodiments, the administration of the O₂ carrierpolypeptide and/or the administration of the chemotherapy is repeated.In some embodiments, the immunotherapy is one or more of an anticancervaccine, an adoptive immune cell therapy or an agent that targets animmune checkpoint regulator. In some embodiments, the immunotherapytargets one or more of CTLA-4, PD1, PD-L1, or an immune checkpointregulator. In some embodiments, the adoptive immune therapy is achimeric antigen receptor expressing T cell or an engineered TCR-T cell.In some embodiments, the immune therapy is an oncolytic virus or aBispecific T cell Engager (BiTE). In some embodiments, theadministration of the O₂ carrier polypeptide and/or the administrationof the immunotherapy is repeated.

In some embodiments of the above embodiments, the O₂ carrier polypeptideis in a pharmaceutical composition. In some embodiments, thepharmaceutical composition further comprises a pharmaceuticallyacceptable carrier. In some embodiments of any of the above embodiments,the O₂ carrier polypeptide is an H-NOX protein.

In some aspects, the invention provides methods for modulating tumorimmunity in an individual with a tumor comprising administering to theindividual an effective amount of an H-NOX protein. In some embodiments,the invention provides methods for enhancing an immune response to thetumor. In some embodiments, the invention provides methods forincreasing leucocyte infiltration to a tumor in an individual comprisingadministering to the individual an effective amount of an H-NOX protein.In some embodiments, the invention provides methods for increasinglymphocyte infiltration to a tumor in an individual comprisingadministering to the individual an effective amount of an H-NOX protein.In some embodiments, the increase in lymphocyte infiltration to thetumor comprises an increase in infiltration of one or more of CD4 cells,CD8 cells, or NK cells. In some embodiments, the increase in lymphocyteinfiltration to the tumor is accompanied by inhibition of one or more ofTreg cells, tumor associated macrophages or myeloid derived suppressorcells in the tumor. In some embodiments, the increase in lymphocyteinfiltration to the tumor is accompanied by an increase in MHC1expression on the tumor cells. In some embodiments, the modulating oftumor immunity comprises increasing antigen processing. In someembodiments, the modulating of tumor immunity comprises increasinglymphocyte activation. In some embodiments, the modulating of tumorimmunity comprises increasing the presentation capabilities of dendriticcells (DC).

In some embodiments, the invention provides methods for decreasingexpression of hypoxia inducible factor 1α (HIF-1α) and/or hypoxiainducible factor 2α (HIF-2α) in a tumor in an individual comprisingadministering to the individual an effective amount of an H-NOX protein.In some embodiments, the invention provides methods for decreasingexpression of programmed death ligand-1 (PD-L1) in a tumor in anindividual comprising administering to the individual an effectiveamount of an H-NOX protein. In some embodiments, the invention providesmethods for decreasing expression of A2A adenosine receptor (A2AR) in atumor in an individual comprising administering to the individual aneffective amount of an H-NOX protein.

In some embodiments of the above embodiments, the tumor is a braintumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, atumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, anovarian tumor, a colorectal tumor, an anal tumor, a liver tumor, ahepatocellular carcinoma, a stomach tumor, a testicular tumor, anendometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin'slymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinaltumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidneytumor, breast tumor, a multiple myeloma tumor, a sarcoma, or a squamouscell tumor.

In some aspects, the invention provides methods for treating cancer inan individual comprising administering to the individual an effectiveamount of an H-NOX protein. In some embodiments, the cancer is braincancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, melanoma, lung cancer, uterine cancer,ovarian cancer, colorectal cancer, anal cancer, liver cancer,hepatocellular carcinoma, stomach cancer, testicular cancer, endometrialcancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma,esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer,bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcomaor squamous cell cancer.

In some embodiments of the above aspects and embodiments, the individualis a mammal. In further embodiments, the mammal is a human (e.g., ahuman patient). In other embodiments, the mammal is a pet, a laboratoryresearch animal, or a farm animal. In some embodiments, the pet,research animal or farm animal is a dog, a cat, a horse, a monkey, arabbit, a rat, a mouse, a guinea pig, a hamster, a pig, or a cow.

In some embodiments of the above aspects and embodiments, the H-NOXprotein is administered by intravenous, intra-arterial, intratumoral,intravesicular, inhalation, intraperitoneal, intrapulmonary,intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular,intrathecal, or transdermal administration. In some embodiments,administration of the H-NOX protein is repeated. In some embodiments,administration of the H-NOX protein is repeated daily or twice a dayfrom about 4 weeks to about 8 weeks. In some embodiments, the H-NOXprotein is administered every four, every 8, every 12, every 24 hours,or every 48 hours for a period of about one to about 10 days. In someembodiments, the H-NOX protein is administered as a bolus. In otherembodiments, the H-NOX protein is administered by infusion. In someembodiments, the H-NOX protein is infused in the individual for about 15minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours,about 6 hours, about 12 hours, about 24 hours or more than 24 hours.

In some embodiments, the invention provides methods to modulate tumorimmunity or to treat cancer in an individual wherein an H-NOX protein isadministered in combination with radiation therapy. In some embodiments,the radiation therapy is administered to the individual 1, 2, 3, 4, 5,6, 8, 10, 12, 14, 16, 18, 20 or 24 hours after the H-NOX protein isadministered. In some embodiments, the radiation is X-radiation. In someembodiments, the X-radiation is administered at about 0.5 gray to about75 gray. In some embodiments, the administration of the H-NOX proteinand/or the administration of the radiation is repeated. In someembodiments, the administration is repeated more than about any of two,three, four times, five times, ten times, 15 times, 20 times, 25 times,30 times or 40 times. In some embodiments, the administration isrepeated after one week, two weeks, three weeks, or four weeks or more.

In some embodiments, the invention provides methods to modulate tumorimmunity or to treat cancer in an individual wherein an H-NOX protein isadministered in combination with chemotherapy or immunotherapy. In someembodiments, the chemotherapy comprises a cytotoxin. In someembodiments, the administration of the H-NOX protein and/or theadministration of the chemotherapy is repeated. In some embodiments, theimmunotherapy is one or more of an anticancer vaccine, an adoptiveimmune cell therapy or an agent that targets an immune checkpointregulator. In some embodiments, the immunotherapy targets one or more ofCTLA-4, PD1, PD-L1, or an immune checkpoint regulator. In someembodiments, the adoptive immude therapy is a chimeric antigen receptorexpressing T cell or an engineered TCR-T cell. In some embodiments, theimmune therapy is an oncolytic virus or a Bispecific T cell Engager(BiTE). In some embodiments, the administration of the H-NOX proteinand/or the administration of the immunotherapy is repeated.

In some embodiments of the above aspects and embodiments, the H-NOXprotein is a T. tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a H.sapiens β1, a R. norvegicus β1, a C. lupus H-NOX, a D. melangaster β1, aD. melangaster CG14885-PA, a C. elegans GCY-35, a N. punctiforme H-NOX,C. crescentus H-NOX, a S. oneidensis H-NOX, or C. acetobutylicum H-NOX.In some embodiments, the H-NOX protein comprises a H-NOX domaincorresponding to the H-NOX domain of T. tengcongensis set forth in SEQID NO:2.

In some embodiments, the H-NOX comprises one or more distal pocketmutations. In some embodiments, the distal pocket mutation is an aminoacid substitution at a site corresponding to L144 of T. tengcongensisH-NOX. In some embodiments, the H-NOX is a T. tengcongensis H-NOXcomprising an amino acid substitution at position 144. In someembodiments, the amino acid substitution at position 144 is an L144Fsubstitution.

In some embodiments, the H-NOX protein is a polymeric H-NOX protein. Insome embodiments, the polymeric H-NOX protein comprises monomers,wherein the monomers comprise an H-NOX domain and a polymerizationdomain. In some embodiments, the H-NOX domain is covalently linked tothe polymerization domain. In some embodiments, the polymeric H-NOXprotein is a trimeric H-NOX protein. In some embodiments, the trimericH-NOX protein comprises one or more trimerization domains. In someembodiments, the trimeric H-NOX protein comprises three monomers,wherein the monomers comprise an H-NOX domain and a trimerizationdomain, wherein the trimerization domain is a bacteriophage T4trimerization domain. In some embodiments, the trimerization domain is afoldon domain. In some embodiments, the foldon domain comprises theamino acid sequence of SEQ ID NO:4.

In some embodiments, the H-NOX protein is fused to an Fc domain of animmunoglobulin. In some embodiments, the H-NOX protein is covalentlybound to polyethylene glycol.

In some embodiments, the O₂ dissociation constant of the H-NOX proteinis within 2 orders of magnitude of that of hemoglobin, and wherein theNO reactivity of the H-NOX protein is at least 10-fold lower than thatof hemoglobin. In some embodiments, the O₂ dissociation constant of thepolymeric H-NOX protein is between about 1 nM and about 1000 nM at 20°C. In some embodiments, the O₂ dissociation constant of the H-NOXprotein is between about 1 μM and about 10 μM at 20° C. In someembodiments, the O₂ dissociation constant of the H-NOX protein isbetween about 10 μM and about 50 μM at 20° C. In some embodiments, theNO reactivity of the H-NOX protein is less than about 700 s⁻¹ at 20° C.In some embodiments, the NO reactivity of the H-NOX protein is at least100-fold lower than that of hemoglobin. In some embodiments, the NOreactivity of the H-NOX protein is at least 1,000-fold lower than thatof hemoglobin. In some embodiments, the k_(off) for oxygen of the H-NOXprotein is less than or equal to about 0.65 s⁻¹ at 20° C. In someembodiments, the k_(off) for oxygen of the H-NOX protein is betweenabout 0.21 s⁻¹ and about 0.65 s⁻¹ at 20° C. In some embodiments, thek_(off) for oxygen of the H-NOX protein is between about 1.35 s⁻¹ andabout 2.9 s⁻¹ at 20° C. In some embodiments, the rate of hemeautoxidation of the H-NOX protein is less than about 1 h⁻¹ at 37° C.

In some embodiments of the above embodiments, the H-NOX protein is in apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier.

In some aspects the invention provides the use of an O₂ carrier proteinfor modulating tumor immunity in an individual. In some embodiments, themodulating tumor immunity comprises enhancing an immune response to thetumor. In some embodiments, the invention provides the use of an O₂carrier polypeptide for increasing leucocyte infiltration to a tumor inan individual. In some embodiments, the invention provides the use of anO₂ carrier polypeptide for increasing lymphocyte infiltration to a tumorin an individual. In some embodiments, the increase in lymphocyteinfiltration to the tumor comprises an increase in infiltration of oneor more of CD4 cells, CD8 cells, or NK cells. In some embodiments, theincrease in lymphocyte infiltration to the tumor is accompanied byinhibition of one or more of Treg cells, tumor associated macrophages ormyeloid derived suppressor cells in the tumor. In some embodiments, theincrease in leucocyte infiltration to the tumor is accompanied by anincrease in MHC1 expression on the tumor cells. In some embodiments, theincrease in lymphocyte infiltration to the tumor is accompanied by anincrease in MHC1 expression on the tumor cells.

In some embodiments, the invention provides the use of an O₂ carrierpolypeptide for decreasing expression of HIF-1α and/or HIF-2α in a tumorin an individual. In some embodiments, the invention provides the use ofan O₂ carrier polypeptide for decreasing expression of PD-L1 in a tumorin an individual. In some embodiments, the invention provides the use ofan O₂ carrier polypeptide for decreasing expression of A2AR in a tumorin an individual.

In some embodiments of the above uses, the tumor is a brain tumor, aglioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, a tumor ofthe head or neck, a melanoma, a lung tumor, a uterine tumor, an ovariantumor, a colorectal tumor, an anal tumor, a liver tumor, ahepatocellular carcinoma, a stomach tumor, a testicular tumor, anendometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin'slymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinaltumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidneytumor, a breast tumor, a multiple myeloma tumor, a sarcoma, or asquamous cell tumor.

In some embodiments, the invention provides the use of an O₂ carrierprotein for treating cancer in an individual. In some embodiments, thecancer is brain cancer, glioblastoma, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, melanoma, lung cancer, uterinecancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer,hepatocellular carcinoma, stomach cancer, testicular cancer, endometrialcancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma,esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer,bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma,or squamous cell cancer.

In some embodiments of the above uses, the individual is a mammal. Insome embodiments, the mammal is a human.

In some embodiments of the above uses, the O₂ carrier polypeptide is anH-NOX protein. In some embodiments, the H-NOX protein is a T.tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a H. sapiens β1, a R.norvegicus β1, a C. lupus H-NOX, a D. melangaster (1, a D. melangasterCG14885-PA, a C. elegans GCY-35, a N. punctiforme H-NOX, C. crescentusH-NOX, a S. oneidensis H-NOX, or C. acetobutylicum H-NOX. In someembodiments, the H-NOX protein comprises a H-NOX domain corresponding tothe H-NOX domain of T. tengcongensis set forth in SEQ ID NO:2. In someembodiments, the H-NOX comprises one or more distal pocket mutations. Insome embodiments, the distal pocket mutation is an amino acidsubstitution at a site corresponding to L144 of T. tengcongensis H-NOX.In some embodiments, the H-NOX is a T. tengcongensis H-NOX comprising anamino acid substitution at position 144. In some embodiments, the aminoacid substitution at position 144 is an L144F substitution.

In some embodiments, the H-NOX protein is a polymeric H-NOX protein. Insome embodiments, the polymeric H-NOX protein comprises monomers,wherein the monomers comprise an H-NOX domain and a polymerizationdomain. In some embodiments, the H-NOX domain is covalently linked tothe polymerization domain. In some embodiments, the polymeric H-NOXprotein is a trimeric H-NOX protein. In some embodiments, the trimericH-NOX protein comprises one or more trimerization domains. In someembodiments, the trimeric H-NOX protein comprises three monomers,wherein the monomers comprise an H-NOX domain and a trimerizationdomain, wherein the trimerization domain is a bacteriophage T4trimerization domain. In some embodiments, the trimerization domain is afoldon domain. In some embodiments, the foldon domain comprises theamino acid sequence of SEQ ID NO:4.

In some embodiments, the H-NOX protein is fused to an Fc domain of animmunoglobulin. In some embodiments, the H-NOX protein is covalentlybound to polyethylene glycol.

In some aspects, the invention provides kits for modulating tumorimmunity in an individual comprising an O₂ carrier protein for use inthe methods described herein. In some embodiments, the kit furthercomprises one or more of a vial, a vessel, an ampule, a bottle, a jars,or flexible packaging. In some embodiments, the kit further comprisesone or more buffer. In some embodiments, the kit further comprisesinstructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows a model of the major immunosuppressive pathwayspromoted by hypoxia (FIG. 1A) and the points of therapeutic interventionthat may be exerted by O₂ carrier polypeptide treatment (FIG. 1B).

FIGS. 2A-2C show tumor oxygenation after single bolus dose of PEGylatedtrimer Tt H-NOX L144F assessed by pimonidazole and HIF-1 ELISA. FIG. 2Ashows pimonidazole levels measured by competitive ELISA. FIG. 2B showsHIF-1α levels measured by sandwich ELISA. Graphs show quantification ofpimonidazole and HIF-1α signals after PEGylated trimer Tt H-NOX L144Fadministration. Mean values+/−SEM. ***p<0.001, **p<0.01 by One way ANOVAand Bonferroni's post-hoc tests. FIG. 2C shows assessment of tumors forthe accumulation of PEGylated trimer Tt H-NOX L144F by sandwich H-NOXELISA and results expressed per gram of tumor tissue.

FIGS. 3A-3D show direct measurements of tumor tissue oxygenationfollowing PEGylated trimer Tt H-NOX L144F administration. Tumors weretreated either with PEGylated trimer TL H-NOX L144F (FIG. 3A),non-functional control Tt H-NOX protein (FIG. 3C), or with 100% oxygenstarting at pO₂=0.44 mmHg (FIG. 3B) with 100% oxygen starting at pO₂=5mmHg (FIG. 3D).

FIG. 4 shows enhancement of radiation efficacy following PEGylatedtrimer Tt H-NOX L144F treatment of mice bearing H460 tumors. Micebearing H460 subcutaneous xenograft tumors (150-300 mm³) were eitherpre-treated with PEGylated trimer Tt H-NOX L144F or treated with 10 Gyalone, irradiated, tumors extracted and processed for clonogenic assay.Cell numbers were counted 7 days later in triplicate samples from eachtumor. Each dot on the graph represents average surviving fraction forone tumor.

FIGS. 5A-5C show PEGylated trimer Tt H-NOX L144F downregulates HIF-1αtargets involved in immunosuppression. Mice bearing H460 subcutaneousxenograft tumors (150-300 mm³) were either pre-treated with PEGylatedtrimer Tt H-NOX L144For treated with vehicle alone, and harvested forqRT-PCR analysis. FIG. 5A shows expression of VEGF. FIG. 5B showsexpression of GLUT1. FIG. 5C shows expression of PD-L1.

FIG. 6A shows the nucleic acid (SEQ ID NO:5) and amino acid sequence(SEQ ID NO:6) of the foldon domain of bacteriophage T4 fibritin fused tothe C-terminus of a Thermoanaerobacter tengcongensis L144F H-NOXsequence and including the His6 tag. FIG. 6B shows the nucleic acid (SEQID NO:7) and amino acid sequence (SEQ ID NO:8) of the L144F H-NOX-foldonmonomer without a His6 tag.

FIGS. 7A-7C show representative images of tumor hypoxia and T cellinfiltration is B16F10 subcutaneous tumors (FIG. 7A), CT26 subcutaneoustumors (FIG. 7B) and GL261 intracranial tumors (FIG. 7C). Hypoxia (toppanels) and T cell infiltration (middle panels) is shown byimmunohistochemistry. Bottom panels show results of quantitativeanalysis of multiple tumor sections. Significantly fewer CD4 and CD8 Tcells infiltrate the hypoxic regions of tumors.

FIG. 8 shows quantification of CD8 T cells in hypoxic areas of tumorsafter H-NOX treatment (OMX) or vehicle control treatment (Veh).Representative images are shown. Hypoxic areas were labeled withpimondazole by immunohistochemical analysis. Following OMX treatmentthere is an increase in CD4 (data not shown) and CD8 T cell infiltrationinto regions of tumors that were hypoxic prior to OMX administration.

FIGS. 9A and 9B show quantification of T cells in normoxic and hypoxicareas of tumors after H-NOX treatment (OMX) or vehicle control treatment(Veh). Both CD4 and CD8 T cells were evaluated. Tumor areas evaluatedincluded areas on the periphery of the tumor and in the tumor center.Results of quantitative image analysis of multiple sections are shown inFIG. 9A and representative images in FIG. 9B. Hypoxic areas werelabelled using immunohistochemical analysis of carbonic anhydrase IX(CAIX) expression. Following OMX treatment, there is an increase in CD4and CD8 T cell infiltration into regions of tumors that were hypoxicprior to OMX administration.

FIG. 10 shows the results of immunohistochemistry for hypoxia(pimondazole) and CD3 vessels is GL261 tumor model.

FIG. 11 shows immunohistochemical analysis of H-NOX tumor penetration,tumor hypoxia and CD8 T cell localization in canine oral melanomatumors. Tissues were stained with hematoxylin and eosin (H&E), DNAinterchelating dye (DAPI) and with anti-H-NOX (OMX), -carbonic anhydraseIX (CAIX) and -CD8 antibodies to assess CD8 lymphocyte localization intumor regions that were hypoxic prior to H-NOX (OMX) treatment. Imagesreveal CD8 positive T cells localized throughout regions of the tumorthat were hypoxic prior to H-NOX (OMX) treatment (CAIX positive).

FIGS. 12A-12K show that larger tumor size correlates with enhancedhypoxia and reduced lymphocyte infiltration in subcutaneous 4T1-Lucsyngeneic mouse tumors. FIG. 12A shows tumor volumes on day 10 and day14 post-implant. FIG. 12B shows fraction of lymphocytes within theviable cell population. FIG. 12C shows the absolute lymphocyte cellnumbers within the viable population. FIG. 12D shows a negativecorrelation between tumor volume and percentage lymphocytes. FIG. 12Eshows a positive correlation between tumor volume and percentagehypoxia. FIG. 12F shows a negative correlation between percentagehypoxia and percentage lymphocytes. FIG. 12G shows a negativecorrelation between tumor volume and percentage CD3-positive T cells.FIG. 12H shows a negative correlation between tumor volume andpercentage CD4-positive T cells. FIG. 12I shows a negative correlationbetween tumor volume and percentage CD8-positive T cells. FIG. 12J showsa negative correlation between tumor volume and percentageCD3-CD4-double-positive T cells. FIG. 12K shows a negative correlationbetween tumor volume and percentage CD3-CD8-double-positive T cells.

FIGS. 13A-13F shows that hypoxic tumor regions are immunosuppressive andexhibit reduced T cell infiltration in subcutaneous 4T1-Luc syngeneicmouse tumors. Immunofluorescence staining of tumor region #1 for (FIG.13A) pimonidazole-positive hypoxic areas and (FIG. 13B) CD8-positive Tcells, counterstained with (FIG. 13C) DAPI to highlight nuclei.Immunofluorescence staining of tumor region #2 for (FIG. 13D)pimonidazole-positive hypoxic areas and (FIG. 13E) CD4-positive T cells,counterstained with (FIG. 13F) DAPI to highlight nuclei.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating cancer in anindividual comprising administering to the individual an effectiveamount of an O₂ carrier polypeptide such as an H-NOX protein. In certainaspects, the invention provides methods for modulating hypoxia-mediatedtumor immunity in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide, such as anH-NOX protein. The O₂ carrier polypeptide is delivered to the tumorwhere it enhances an immune response to the tumor. Enhancement of animmune response to the tumor may be mediated by targeting hypoxiainducible factor 1α (HIF-1α)-mediated pathways of tumor immunity and/ornon-HIF-1α-mediated pathways of tumor immunity. In some aspects, theinvention provides methods for increasing lymphocyte infiltration to atumor in an individual comprising administering to the individual aneffective amount of an O₂ carrier polypeptide. In some embodiments, theincrease in lymphocyte infiltration to the tumor comprises an increasein infiltration of one or more of CD4 cells, CD8 cells, or NK cells. Insome embodiments, the increase in lymphocyte infiltration to the tumoris accompanied by inhibition of one or more of Treg cells, tumorassociated macrophages or myeloid derived suppressor cells in the tumor.In some embodiments, the increase in lymphocyte infiltration to thetumor is accompanied by an increase in MHC1 expression on the tumorcells. In some embodiments, the invention provides methods fordecreasing expression of hypoxia inducible factor 1α (HIF-1α) in a tumorin an individual comprising administering to the individual an effectiveamount of an O₂ carrier polypeptide (e.g., an H-NOX protein). In someembodiments, the invention provides methods for decreasing expression ofprogrammed death ligand-1 (PD-L1) in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide (e.g., an H-NOX protein). In some embodiments, the inventionprovides methods for decreasing expression of A2A adenosine receptor(A2AR) in a tumor in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide (e.g., anH-NOX protein).

Definitions

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. One of skill in the art will alsoappreciate that any methods and materials similar or equivalent to thosedescribed herein can also be used to practice or test the invention.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and polymers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification. As used herein, aprotein may include two or more subunits, covalently or non-covalentlyassociated; for example, a protein may include two or more associatedmonomers.

The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidesthat comprise the nucleic acid molecule or polynucleotide.

As used herein, the term “hypoxia inducible factor” or “HIF” refers to afamily of transcription factor that respond to decreases in oxygen, orhypoxia, in the cellular environment. Members of the human HIF familyinclude HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF3α, HIF3β. HIF-1 functions asa master regulator of homeostatic responses to hypoxia by activatingtranscription of many genes, including those involved in energymetabolism, angiogenesis, apoptosis, and other genes whose proteinproducts increase oxygen delivery or facilitate metabolic adaptation tohypoxia. HIF-1 plays a role in embryonic vascularization, tumorangiogenesis and pathophysiology of ischemic disease. Humanhypoxia-inducible factor 1, alpha subunit or human HIF-1α interacts witha number of polypeptides including but not limited to ARNTL, ARNT,CREBB, EP300, HIF-1AN, Mdm2, NR4A, p53, PSMA7, STAT3, UBC, VH and pVHL.Human HIF-1α is encoded by the HIF-1A gene. The amino acid sequence ofhuman HIF-1α is provided by GenBank Accession no. NP_001230013 and thenucleotide sequence of human HIF-1α mRNA is provided by GenBankAccession No. NM_001243084. The amino acid sequence of mouse HIF-1α isprovided by GenBank Accession no. NP_010431 and the nucleotide sequenceof mouse HIF-1α mRNA is provided by GenBank Accession No. NM_034561.

As used herein, “programmed death-ligand 1” or “PD-L1” refers to atransmembrane protein that is part of an immune checkpoint pathway thatplays a role in suppressing the immune system. Interaction of PDL1 withthe PD1 receptor or the B7.1 receptor inhibits T cell receptor-mediatedactivation of IL-2 and T cell proliferation. Human PD-L1 is encoded bythe CD274 gene. The amino acid sequence of human PD-L1 is provided byGenBank Accession no. NP_001254635 and the nucleotide sequence of humanPD-L1 mRNA is provided by GenBank Accession No. NM_001267706. The aminoacid sequence of mouse PD-L1 is provided by GenBank Accession no.NP_021893 and the nucleotide sequence of mouse PD-L1 mRNA is provided byGenBank Accession No. NM_068693.

As used herein, an “adenosine A2A receptor” or “A2AR” refers to areceptor of the G protein-coupled receptor superfamily. A2AR is areceptor for adenosine that plays a role in oxygen consumption and isthought to play a role in suppressing overreactive immune cells by wayor increased levels of cAMP. Human A2AR is encoded by ADORA2A the gene.The amino acid sequence of human A2AR is provided by GenBank Accessionno. NP_000666 and the nucleotide sequence of human A2AR mRNA is providedby GenBank Accession No. NM_000675. The amino acid sequence of mouseA2AR is provided by GenBank Accession no. NP_033760 and the nucleotidesequence of mouse A2AR mRNA is provided by GenBank Accession No. NM09630.

As used herein, an “H-NOX protein” means a protein that has an H-NOXdomain (named for Heme-Nitric oxide and OXygen binding domain). An H-NOXprotein may or may not contain one or more other domains in addition tothe H-NOX domain. In some examples, an H-NOX protein does not comprise aguanylyl cyclase domain. An H-NOX protein may or may not comprise apolymerization domain.

As used herein, a “polymeric H-NOX protein” is an H-NOX proteincomprising two or more H-NOX domains. The H-NOX domains may becovalently or non-covalently associated.

As used herein, an “H-NOX domain” is all or a portion of a protein thatbinds nitric oxide and/or oxygen by way of heme. The H-NOX domain maycomprise heme or may be found as an apoproprotein that is capable ofbinding heme. In some examples, an H-NOX domain includes sixalpha-helices, followed by two beta-strands, followed by onealpha-helix, followed by two beta strands. In some examples, an H-NOXdomain corresponds to the H-NOX domain of Thermoanaerobactertengcongensis H-NOX set forth in SEQ ID NO:2. For example, the H-NOXdomain may be at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 99% identical to the H-NOX domain ofThermoanaerobacter tengcongensis H-NOX set forth in SEQ ID NO:2. In someembodiments, the H-NOX domain may be 10%-20%, 20%-30%, 30%-40%, 40%-50%,50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% or 100% identicalto the H-NOX domain of Thermoanaerobacter tengcongensis H-NOX set forthin SEQ ID NO:2.

As used herein, a “polymerization domain” is a domain (e.g. apolypeptide domain) that promotes the association of monomeric moietiesto form a polymeric structure. For example, a polymerization domain maypromote the association of monomeric H-NOX domains to generate apolymeric H-NOX protein. An exemplary polymerization domain is thefoldon domain of T4 bacteriophage, which promotes the formation oftrimeric polypeptides. Other examples of polymerization domains include,but are not limited to, Arc, POZ, coiled coil domains (including GCN4,leucine zippers, Velcro), uteroglobin, collagen, 3-stranded coiled colis(matrilin-1), thrombosporins, TRPV1-C, P53, Mnt, avadin, streptavidin,Bcr-Abl, COMP, verotoxin subunit B, CamKII, RCK, and domains from Nethylmaleimide-sensitive fusion protein, STM3548, KaiC, TyrR, Hcp1,CcmK4, GP41, anthrax protective antigen, aerolysin, a-hemolysin,C4b-binding protein, Mi-CK, arylsurfatase A, and viral capsid proteins.

As used herein, an “amino acid linker sequence” or an “amino acid spacersequence” is a short polypeptide sequence that may be used to link twodomains of a protein. In some embodiments, the amino acid linkersequence is one, two, three, four, five, six, seven, eight, nine, ten ormore than ten amino acids in length. Exemplary amino acid linkersequences include but are not limited to a Gly-Ser-Gly sequence and anArg-Gly-Ser sequence.

As used herein, a “His₆ tag” refers to a peptide comprising six Hisresidues attached to a polypeptide. A His₆ tag may be used to facilitateprotein purification; for example, using chromatography specific for theHis₆ tag. Following purification, the His₆ tag may be cleaved using anexopeptidase.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values such that one of skill in the art would consider thedifference between the two values to be of statistical significancewithin the context of the biological characteristic measured by saidvalues. In some embodiments, the two substantially different numericvalues differ by greater than about any one of 10%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, or 90%. In some embodiment, the twosubstantially different numeric values differ by about any one of10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%,90%-95%, 95%-99% or 100%.

A “native sequence” polypeptide comprises a polypeptide having the sameamino acid sequence as a polypeptide found in nature. Thus, a nativesequence polypeptide can have the amino acid sequence of naturallyoccurring polypeptide from any organism. Such native sequencepolypeptide can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence” polypeptidespecifically encompasses naturally occurring truncated or secreted formsof the polypeptide (e.g., an extracellular domain sequence), naturallyoccurring variant forms (e.g., alternatively spliced forms) andnaturally occurring allelic variants of the polypeptide.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about any one of 80%, 90% or 95% amino acid sequence identitywith the native sequence polypeptide. In some embodiments, a variantwill have about any one of 80%-90%, 90%-95% or 95%-99% amino acidsequence identity with the native sequence polypeptide.

As used herein, a “mutant protein” means a protein with one or moremutations compared to a protein occurring in nature. In one embodiment,the mutant protein has a sequence that differs from that of all proteinsoccurring in nature. In various embodiments, the amino acid sequence ofthe mutant protein is at least about any of 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, 95, 97, 98, 99, or 99.5% identical to that of thecorresponding region of a protein occurring in nature. In someembodiments, the amino acid sequence of the mutant protein is at leastabout any of 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%,70%-80%, 80%-90%, 90%-95%, 95%-99% or 100% identical to that of thecorresponding region of a protein occurring in nature. In someembodiments, the mutant protein is a protein fragment that contains atleast about any of 25, 50, 75, 100, 150, 200, 300, or 400 contiguousamino acids from a full-length protein. In some embodiments, the mutantprotein is a protein fragment that contains about any of 25-50, 50-75,75-100, 100-150, 150-200, 200-300, or 300-400 contiguous amino acidsfrom a full-length protein. Sequence identity can be measured, forexample, using sequence analysis software with the default parametersspecified therein (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). This software programmatches similar sequences by assigning degrees of homology to variousamino acids replacements, deletions, and other modifications.

As used herein, a “mutation” means an alteration in a reference nucleicacid or amino acid sequence occurring in nature. Exemplary nucleic acidmutations include an insertion, deletion, frameshift mutation, silentmutation, nonsense mutation, or missense mutation. In some embodiments,the nucleic acid mutation is not a silent mutation. Exemplary proteinmutations include the insertion of one or more amino acids (e.g., theinsertion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), the deletion ofone or more amino acids (e.g., a deletion of N-terminal, C-terminal,and/or internal residues, such as the deletion of at least about any of5, 10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or adeletion of about any of 5-10, 10-15, 15-25, 25-50, 50-75, 75-100,100-150, 150-200, 200-300, or 300-400 amino acids), the replacement ofone or more amino acids (e.g., the replacement of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids), or combinations of two or more of theforegoing. The nomenclature used in referring to a particular amino acidmutation first identifies the wild-type amino acid, followed by theresidue number and finally the substitute amino acid. For example, Y140Lmeans that tyrosine has been replaced by a leucine at residue number140. Likewise, a variant H-NOX protein may be referred to by the aminoacid variations of the H-NOX protein. For example, a T. tengcongensisY140L H-NOX protein refers to a T. tengcongensis H-NOX protein in whichthe tyrosine residue at position number 140 has been replaced by aleucine residue and a T. tengcongensis W9F/Y140L H-NOX protein refers toa T. tengcongensis H-NOX protein in which the tryptophan residue atposition 9 has been replaced by a phenylalanine residue and the tyrosineresidue at position number 140 has been replaced by a leucine residue.

An “evolutionary conserved mutation” is the replacement of an amino acidin one protein by an amino acid in the corresponding position of anotherprotein in the same protein family.

As used herein, “derived from” refers to the source of the protein intowhich one or more mutations is introduced. For example, a protein thatis “derived from a mammalian protein” refers to protein of interest thatresults from introducing one or more mutations into the sequence of awild-type (i.e., a sequence occurring in nature) mammalian protein.

As used herein, “Percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, a “k_(off)” refers to a dissociation rate, such as therate of release of O₂ or NO from a protein. A lower numerical lowerk_(off) indicates a slower rate of dissociation.

As used herein, “k_(on)” refers to an association rate, such as the rateof binding of O₂ or NO to a protein. A lower numerical lower k_(on)indicates a slower rate of association.

As used herein, “dissociation constant” refers to a “kineticdissociation constant” or a “calculated dissociation constant.” A“kinetic dissociation constant” or “K_(D)” is a ratio of kineticoff-rate (k_(off)) to kinetic on-rate (k_(on)), such as a K_(D) valuedetermined as an absolute value using standard methods (e.g., standardspectroscopic, stopped-flow, or flash-photolysis methods) includingmethods known to the skilled artisan and/or described herein.“Calculated dissociation constant” or “calculated K_(D)” refers to anapproximation of the kinetic dissociation constant based on a measuredk_(off). A value for the k_(on) is derived via the correlation betweenkinetic K_(D) and k_(off) as described herein.

As used herein, “oxygen affinity” is a qualitative term that refers tothe strength of oxygen binding to the heme moiety of a protein. Thisaffinity is affected by both the k_(off) and k_(on) for oxygen. Anumerically lower oxygen K_(D) value means a higher affinity.

As used herein, “NO affinity” is a qualitative term that refers to thestrength of NO binding to a protein (such as binding to a heme group orto an oxygen bound to a heme group associated with a protein). Thisaffinity is affected by both the k_(off) and k_(on) for NO. Anumerically lower NO K_(D) value means a higher affinity.

As used herein, “NO stability” refers to the stability or resistance ofa protein to oxidation by NO in the presence of oxygen. For example, theability of the protein to not be oxidized when bound to NO in thepresence of oxygen is indicative of the protein's NO stability. In someembodiments, less than about any of 50, 40, 30, 10, or 5% of an H-NOXprotein is oxidized after incubation for about any of 1, 2, 4, 6, 8, 10,15, or 20 hours at 20° C.

As used herein, “NO reactivity” refers to the rate at which iron in theheme of a heme-binding protein is oxidized by NO in the presence ofoxygen. A lower numerical value for NO reactivity in units of s⁻¹indicates a lower NO reactivity

As used herein, an “autoxidation rate” refers to the rate at which ironin the heme of a heme-binding protein is autoxidized. A lower numericalautoxidation rate in units of s⁻¹ indicates a lower autoxidation rate.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Exemplary prokaryotic cellsinclude bacterial cells; for example, E. coli cells.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, e.g., in the case of anRNA polynucleotide. Thus, a DNA polynucleotide that is contained in avector inside a host cell may be referred to as “isolated”.

The terms “individual” or “subject” are used interchangeably herein torefer to an animal; for example a mammal. In some embodiments, methodsof treating mammals, including, but not limited to, humans, rodents,simians, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder.

A “disease” or “disorder” as used herein refers to a condition wheretreatment is needed.

The term “cancer” refers to a malignant proliferative disorderassociated with uncontrolled cell proliferation, unrestrained cellgrowth, and decreased cell death via apoptosis.

The term “tumor” is used herein to refer to a group of cells thatexhibit abnormally high levels of proliferation and growth. A tumor maybe benign, pre-malignant, or malignant; malignant tumor cells arecancerous. Tumor cells may be solid tumor cells or leukemic tumor cells.The term “tumor growth” is used herein to refer to proliferation orgrowth by a cell or cells that comprise a tumor that leads to acorresponding increase in the size of the tumor.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (e.g., notworsening) state of disease, preventing spread (e.g., metastasis) ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methods of theinvention contemplate any one or more of these aspects of treatment.

In the context of cancer, the term “treating” includes any or all of:inhibiting growth of tumor cells or cancer cells, inhibiting replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In certain embodiments, by “reduce”or “inhibit” is meant the ability to cause an overall decrease of 20% orgreater. In another embodiment, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In yet anotherembodiment, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or 99%.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

A “reference” as used herein, refers to any sample, standard, or levelthat is used for comparison purposes. A reference may be obtained from ahealthy and/or non-diseased sample. In some examples, a reference may beobtained from an untreated sample. In some examples, a reference isobtained from a non-diseased on non-treated sample of a subjectindividual. In some examples, a reference is obtained from one or morehealthy individuals who are not the subject or patient.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease.

An “effective amount” of an agent refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A therapeutically effective amountmay be delivered in one or more administrations.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. Such formulations may besterile and essentially free of endotoxins.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

A “sterile” formulation is aseptic or essentially free from livingmicroorganisms and their spores.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent. For example, the two or more therapeutic agentsare administered with a time separation of no more than about 60minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents where the administration of one or moreagent(s) continues after discontinuing the administration of one or moreother agent(s). For example, administration of the two or moretherapeutic agents are administered with a time separation of more thanabout 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1day, 2 days, 3 days, 1 week, 2 weeks, or 1 month.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (e.g., a package orcontainer) or kit comprising at least one reagent, e.g., a medicamentfor treatment of a disease or disorder (e.g., cancer), or a probe forspecifically detecting a biomarker described herein. In certainembodiments, the manufacture or kit is promoted, distributed, or sold asa unit for performing the methods described herein.

H-NOX Proteins Overview of H-NOX Protein Family

Unless otherwise indicated, any wild-type or mutant H-NOX protein can beused in the compositions, kits, and methods as described herein. As usedherein, an “H-NOX protein” means a protein that has an H-NOX domain(named for Heme-Nitric oxide and OXygen binding domain). An H-NOXprotein may or may not contain one or more other domains in addition tothe H-NOX domain. H-NOX proteins are members of a highly-conserved,well-characterized family of hemoproteins (Iyer, L. M. et al. (Feb. 3,2003). BMC Genomics 4(1):5; Karow, D. S. et al. (Aug. 10, 2004).Biochemistry 43(31):10203-10211; Boon, E. M. et al. (2005). Nature Chem.Biol. 1:53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem.Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem.99(4):892-902). H-NOX proteins are also referred to as Pfam 07700proteins or HNOB proteins (Pfam—A database of protein domain familyalignments and Hidden Markov Models, Copyright (C) 1996-2006 The PfamConsortium; GNU LGPL Free Software Foundation, Inc., 59 TemplePlace—Suite 330, Boston, Mass. 02111-1307, USA). In some embodiments, anH-NOX protein has, or is predicted to have, a secondary structure thatincludes six alpha-helices, followed by two beta-strands, followed byone alpha-helix, followed by two beta-strands. An H-NOX protein can bean apoprotein that is capable of binding heme or a holoprotein with hemebound. An H-NOX protein can covalently or non-covalently bind a hemegroup. Some H-NOX proteins bind NO but not Oz, and others bind both NOand O₂. H-NOX domains from facultative aerobes that have been isolatedbind NO but not O₂. H-NOX proteins from obligate aerobic prokaryotes, C.elegans, and D. melanogaster bind NO and O₂. Mammals have two H-NOXproteins: β1 and β2. An alignment of mouse, rat, cow, and human H-NOXsequences shows that these species share >99% identity. In someembodiments, the H-NOX domain of an H-NOX protein or the entire H-NOXprotein is at least about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 95, 97, 98, 99, or 99.5% identical to that of the correspondingregion of a naturally-occurring Thermoanaerobacter tengcongensis H-NOXprotein (e.g. SEQ ID NO:2) or a naturally-occurring sGC protein (e.g., anaturally-occurring sGC β1 protein). In some embodiments, the H-NOXdomain of an H-NOX protein or the entire H-NOX protein is at least aboutany of 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%,90-95%, 95-99, or 99-99.9% identical to that of the corresponding regionof a naturally-occurring Thermoanaerobacter tengcongensis H-NOX protein(e.g. SEQ ID NO:2) or a naturally-occurring sGC protein (e.g., anaturally-occurring sGC 1 protein). As discussed further herein, anH-NOX protein may optionally contain one or more mutations relative tothe corresponding naturally-occurring H-NOX protein. In someembodiments, the H-NOX protein includes one or more domains in additionto the H-NOX domain. In particular embodiments, the H-NOX proteinincludes one or more domains or the entire sequence from anotherprotein. For example, the H-NOX protein may be a fusion protein thatincludes an H-NOX domain and part or all of another protein, such asalbumin (e.g., human serum albumin). In some embodiments, only the H-NOXdomain is present. In some embodiments, the H-NOX protein does notcomprise a guanylyl cyclase domain. In some embodiments, the H-NOXprotein comprises a tag; for example, a His₆ tag.

Polymeric H-NOX Proteins

In some aspects, the invention provides polymeric H-NOX proteinscomprising two or more H-NOX domains. The two or more H-NOX domains maybe covalently linked or noncovalently linked. In some embodiments, thepolymeric H-NOX protein is in the form of a dimer, a trimer, a tetramer,a pentamer, a hexamer, a heptamer, an octomer, a nanomer, or a decamer.In some embodiments, the polymeric H-NOX protein comprises homologousH-NOX domains. In some embodiments, the polymeric H-NOX proteincomprises heterologous H-NOX domains; for example, the H-NOX domains maycomprises amino acid variants of a particular species of H-NOX domain ormay comprise H-NOX domains from different species. In some embodiments,at least one of the H-NOX domains of a polymeric H-NOX protein comprisesa mutation corresponding to an L144F mutation of T. tengcongensis H-NOX.In some embodiments, at least one of the H-NOX domains of a polymericH-NOX protein comprises a mutation corresponding to a W9F/L144F mutationof T. tengcongensis H-NOX. In some embodiments, the polymeric H-NOXproteins comprise one or more polymerization domains. In someembodiments, the polymeric H-NOX protein is a trimeric H-NOX protein. Insome embodiments, the polymeric H-NOX protein comprises at least onetrimerization domain. In some embodiments, the trimeric H-NOX proteincomprises three T. tengcongensis H-NOX domains. In some embodiments thetrimeric H-NOX domain comprises three T. tengcongensis L144F H-NOXdomains (trimeric Tt H-NOX L144F). In some embodiments the trimericH-NOX domain comprises three T. tengcongensis W9F/L144F H-NOX domains

In some aspects of the invention, the polymeric H-NOX protein comprisestwo or more associated monomers. The monomers may be covalently linkedor noncovalently linked. In some embodiments, monomeric subunits of apolymeric H-NOX protein are produced where the monomeric subunitsassociate in vitro or in vivo to form the polymeric H-NOX protein. Insome embodiments, the monomers comprise an H-NOX domain and apolymerization domain. In some embodiments, the polymerization domain iscovalently linked to the H-NOX domain; for example, the C-terminus ofthe H-NOX domain is covalently linked to the N-terminus or theC-terminus of the polymerization domain. In other embodiments, theN-terminus of the H-NOX domain is covalently linked to the N-terminus orthe C-terminus of the polymerization domain. In some embodiments, anamino acid spacer is covalently linked between the H-NOX domain and thepolymerization domain. An “amino acid spacer” and an “amino acid linker”are used interchangeably herein. In some embodiments, at least one ofthe monomeric subunits of a polymeric H-NOX protein comprises a mutationcorresponding to an L144F mutation of T. tengcongensis H-NOX. In someembodiments, at least one of the monomeric subunits of a polymeric H-NOXprotein comprises a mutation corresponding to a W9F/L144F mutation of T.tengcongensis H-NOX. In some embodiments the polymeric H-NOX protein isa trimeric H-NOX protein. In some embodiments, the monomer of a trimericH-NOX protein comprises an H-NOX domain and a foldon domain of T4bacteriophage. In some embodiments, the monomer of a trimeric H-NOXprotein comprises a T. tengcongensis H-NOX domain and a foldon domain.In some embodiments, the monomer of a trimeric H-NOX protein comprises aT. tengcongensis L144F H-NOX domain and a foldon domain. In someembodiments, the monomer of a trimeric H-NOX protein comprises a T.tengcongensis W9F/L144F H-NOX domain and a foldon domain. In someembodiments, the trimer H-NOX protein comprises three monomers, eachmonomer comprising a T. tengcongensis L144F H-NOX domain and a foldondomain. In some embodiments, the H-NOX domain is linked to the foldondomain with an amino acid linker; for example a Gly-Ser-Gly linker. Insome embodiments, at least one H-NOX domain comprises a tag. In someembodiments, at least one H-NOX domain comprises a His₆ tag. In someembodiments, the His₆ tag is linked to the foldon domain with an aminoacid linker; for example an Arg-Gly-Ser linker. In some embodiments, allof the H-NOX domains comprise a His₆ tag. In some embodiments, thetrimeric H-NOX protein comprises the amino acid sequence set forth inSEQ ID NO:6 or SEQ ID NO:8.

The exemplary H-NOX domain from T. tengcongensis is approximately 26.7kDal. In some embodiments, the polymeric H-NOX protein has an atomicmass greater than any of about 50 kDal, 75 kDal, 100 kDal, 125kDal, toabout 150 kDal.

The invention provides polymeric H-NOX proteins that show greateraccumulation in one or more tissues in an individual compared to acorresponding monomeric H-NOX protein comprising a single H-NOX domainfollowing administration of the H-NOX protein to the individual. Acorresponding H-NOX protein refers to a monomeric form of the H-NOXprotein comprising at least one of the H-NOX domains of the polymericH-NOX protein. Tissues of preferential polymeric H-NOX accumulationinclude, but are not limited to tumors and tissue with damagedvasculature. In some embodiments the polymeric H-NOX protein persists ina mammal for at least about 1, 2, 3, 4, 6, 12 or 24 hours followingadministration of the H-NOX protein to the individual. In someembodiments the polymeric H-NOX protein persists in a mammal for about1-2, 2-3, 3-4, 4-6, 6-12 or 12-24 hours following administration of theH-NOX protein to the individual In some embodiments, less than about 10%of the polymeric H-NOX is cleared from mammal by the kidneys within lessthan any of about 1 hour, 2 hours or 3 hours following administration ofthe H-NOX protein to the individual.

Sources of H-NOX Proteins and H-NOX Domains

H-NOX proteins and H-NOX domains from any genus or species can be usedin the compositions, kits, and methods described herein. In variousembodiments, the H-NOX protein or the H-NOX domains of a polymeric H-NOXprotein is a protein or domain from a mammal (e.g., a primate (e.g.,human, monkey, gorilla, ape, lemur, etc), a bovine, an equine, aporcine, a canine, or a feline), an insect, a yeast, or a bacteria or isderived from such a protein. Exemplary mammalian H-NOX proteins includewild-type human and rat soluble guanylate cyclase (such as the 31subunit). Non-limiting examples of H-NOX proteins include wild-typemammalian H-NOX proteins, e.g. H. sapiens, M. musculus, C. familiaris,B. Taurus, C. lupus and R. norvegicus and examples of prokaryoticwild-type H-NOX proteins include T. tengcongensis, V. cholera, V.fischerii, N. punctiforme, D. desulfuricans, L. pneumophila 1, L.pneumophila 2, and C. acetobutvlicum. Examples of H-NOX proteinsincluding their NCBI accession numbers may be found in U.S. Pat. Nos.8,404,631 and 8,404,632, WO 2007/139791 and WO 2007/139767; the contentsof each is incorporated herein by reference in its entirety.

Additional H-NOX proteins, H-NOX domains of polymeric H-NOX proteins,and nucleic acids, which may be suitable for use in the pharmaceuticalcompositions and methods described herein, can be identified usingstandard methods. For example, standard sequence alignment and/orstructure prediction programs can be used to identify additional H-NOXproteins and nucleic acids based on the similarity of their primaryand/or predicted protein secondary structure with that of known H-NOXproteins and nucleic acids. For example, the Pfam database uses definedalignment algorithms and Hidden Markov Models (such as Pfam 21.0) tocategorize proteins into families, such as the H-NOX protein family(Pfam—A database of protein domain family alignments and Hidden MarkovModels, Copyright (C) 1996-2006 The Pfam Consortium; GNU LGPL FreeSoftware Foundation, Inc., 59 Temple Place—Suite 330, Boston, Mass.02111-1307, USA). Standard databases such as the swissprot-trembldatabase (world-wide web at “expasy.org”, Swiss Institute ofBioinformatics Swiss-Prot group CMU—1 rue Michel Servet CH-1211 Geneva4, Switzerland) can also be used to identify members of the H-NOXprotein family. The secondary and/or tertiary structure of an H-NOXprotein can be predicted using the default settings of standardstructure prediction programs, such as PredictProtein (630 West, 168Street, BB217, New York, N.Y. 10032, USA). Alternatively, the actualsecondary and/or tertiary structure of an H-NOX protein can bedetermined using standard methods.

In some embodiments, the H-NOX domain has the same amino acid in thecorresponding position as any of following distal pocket residues in T.tengcongensis H-NOX: Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78,Phe82, Tyr140, Leu144, or any combination of two or more of theforegoing. In some embodiments, the H-NOX domain has a proline or anarginine in a position corresponding to that of Pro115 or Arg135 of T.tengcongensis H-NOX, respectively, based on sequence alignment of theiramino acid sequences. In some embodiments, the H-NOX domain has ahistidine that corresponds to His105 of R. norvegicus β1 H-NOX. In someembodiments, the H-NOX domain has or is predicted to have a secondarystructure that includes six alpha-helices, followed by two beta-strands,followed by one alpha-helix, followed by two beta-strands. Thissecondary structure has been reported for H-NOX proteins.

If desired, a newly identified H-NOX protein or H-NOX domain can betested to determine whether it binds heme using standard methods. Theability of an H-NOX domain to function as an O₂ carrier can be tested bydetermining whether the H-NOX domain binds O₂ using standard methods,such as those described herein. If desired, one or more of the mutationsdescribed herein can be introduced into the H-NOX domain to optimize itscharacteristics as an O₂ carrier. For example, one or more mutations canbe introduced to alter its O₂ dissociation constant, k_(off) for oxygen,rate of heme autoxidation, NO reactivity, NO stability or anycombination of two or more of the foregoing. Standard techniques such asthose described herein can be used to measure these parameters.

Mutant H-NOX Proteins

As discussed further herein, an H-NOX protein or an H-NOX domain of apolymeric H-NOX protein may contain one or more mutations, such as amutation that alters the O₂ dissociation constant, the k_(off) foroxygen, the rate of heme autoxidation, the NO reactivity, the NOstability, or any combination of two or more of the foregoing comparedto that of the corresponding wild-type protein. In some embodiments, theinvention provides a polymeric H-NOX protein comprising one or moreH-NOX domains that may contain one or more mutations, such as a mutationthat alters the O₂ dissociation constant, the k_(off) for oxygen, therate of heme autoxidation, the NO reactivity, the NO stability, or anycombination of two or more of the foregoing compared to that of thecorresponding wild-type protein. Panels of engineered H-NOX domains maybe generated by random mutagenesis followed by empirical screening forrequisite or desired dissociation constants, dissociation rates,NO-reactivity, stability, physio-compatibility, or any combination oftwo or more of the foregoing in view of the teaching provided hereinusing techniques as described herein and, additionally, as known by theskilled artisan. Alternatively, mutagenesis can be selectively targetedto particular regions or residues such as distal pocket residuesapparent from the experimentally determined or predictedthree-dimensional structure of an H-NOX protein (see, for example, Boon,E. M. et al. (2005). Nature Chemical Biology 1:53-59, which is herebyincorporated by reference in its entirety, particularly with respect tothe sequences of wild-type and mutant H-NOX proteins) or evolutionarilyconserved residues identified from sequence alignments (see, forexample, Boon E. M. et al. (2005). Nature Chemical Biology 1:53-59,which is hereby incorporated by reference in its entirety, particularlywith respect to the sequences of wild-type and mutant H-NOX proteins).

In some embodiments of the invention, the mutant H-NOX protein or mutantH-NOX domain of a polymeric H-NOX protein has a sequence that differsfrom that of all H-NOX proteins or domains occurring in nature. Invarious embodiments, the amino acid sequence of the mutant protein is atleast about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97,98, 99, or 99.5% identical to that of the corresponding region of anH-NOX protein occurring in nature. In various embodiments, the aminoacid sequence of the mutant protein is about 10-20%, 20-30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 99.5%identical to that of the corresponding region of an H-NOX proteinoccurring in nature. In some embodiments, the mutant protein is aprotein fragment that contains at least about any of 25, 50, 75, 100,150, 200, 300, or 400 contiguous amino acids from a full-length protein.In some embodiments, the mutant protein is a protein fragment thatcontains 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400contiguous amino acids from a full-length protein. Sequence identity canbe measured, for example, using sequence analysis software with thedefault parameters specified therein (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various amino acids replacements, deletions, and othermodifications.

In some embodiments of the invention, the mutant H-NOX protein or mutantH-NOX domain of a polymeric H-NOX protein comprises the insertion of oneor more amino acids (e.g., the insertion of 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids). In some embodiments of the invention, the mutant H-NOXprotein or mutant H-NOX domain comprises the deletion of one or moreamino acids (e.g., a deletion of N-terminal, C-terminal, and/or internalresidues, such as the deletion of at least about any of 5, 10, 15, 25,50, 75, 100, 150, 200, 300, or more amino acids or a deletion of 5-10,10-15, 15-25, 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or300-400 amino acids). In some embodiments of the invention, the mutantH-NOX protein or mutant H-NOX domain comprises the replacement of one ormore amino acids (e.g., the replacement of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids), or combinations of two or more of the foregoing. Insome embodiments, a mutant protein has at least one amino acidalteration compared to a protein occurring in nature. In someembodiments, a mutant nucleic acid sequence encodes a protein that hasat least one amino acid alteration compared to a protein occurring innature. In some embodiments, the nucleic acid is not a degenerateversion of a nucleic acid occurring in nature that encodes a proteinwith an amino acid sequence identical to a protein occurring in nature.

In some embodiments the mutation in the H-NOX protein or H-NOX domain ofa polymeric H-NOX protein is an evolutionary conserved mutations (alsodenoted class I mutations). Examples of class I mutations are listed inTable 1A. In Table 1A, mutations are numbered/annotated according to thesequence of human β1 H-NOX, but are analogous for all H-NOX sequences.Thus, the corresponding position in any other H-NOX protein can bemutated to the indicated residue. For example, Phe4 of human β1 H-NOXcan be mutated to a tyrosine since other H-NOX proteins have a tyrosinein this position. The corresponding phenylalanine residue can be mutatedto a tyrosine in any other H-NOX protein. In particular embodiments, theone or more mutations are confined to evolutionarily conserved residues.In some embodiments, the one or more mutations may include at least oneevolutionarily conserved mutation and at least one non-evolutionarilyconserved mutation. If desired, these mutant H-NOX proteins aresubjected to empirical screening for NO/O₂ dissociation constants,NO-reactivity, stability, and physio-compatibility in view of theteaching provided herein.

TABLE 1A Exemplary Class I H-NOX mutations targeting evolutionaryconserved residues F4Y Q30G I145Y F4L E33P I145H H7G N61G K151E A8E C78HI157F L9W A109F E183F

In some embodiments, the mutation is a distal pocket mutation, such asmutation of a residue in alpha-helix A, D, E, or G (Pellicena, P. et al.(Aug. 31, 2004). Proc Natl. Acad Sci USA 101(35):12854-12859). Exemplarydistal pocket mutations (also denoted class II mutations) are listed inTable 1B. In Table 1B, mutations are numbered/annotated according to thesequence of human β1 H-NOX, but are analogous for all H-NOX sequences.Because several substitutions provide viable mutations at each recitedresidue, the residue at each indicated position can be changed to anyother naturally or non-naturally-occurring amino acid (denoted “X”).Such mutations can produce H-NOX proteins with a variety of desiredaffinity, stability, and reactivity characteristics.

TABLE 1B Exemplary Class II H-NOX mutations targeting distal pocketresidues V8X M73X I145X L9X F77X I149X F70X C78X

In particular embodiments, the mutation is a heme distal pocketmutation. As described herein, a crucial molecular determinant thatprevents O₂ binding in NO-binding members of the H-NOX family is thelack of a H-bond donor in the distal pocket of the heme. Accordingly, insome embodiments, the mutation alters H-bonding between the H-NOX domainand the ligand within the distal pocket. In some embodiments, themutation disrupts an H-bond donor of the distal pocket and/or impartsreduced O₂ ligand-binding relative to the corresponding wild-type H-NOXdomain. Exemplary distal pocket residues include Thr4, Ile5, Thr8, Trp9,Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of T.tengcongensis H-NOX and the corresponding residues in any other H-NOXprotein. In some embodiments, the H-NOX protein or H-NOX domain of apolymeric H-NOX protein comprises one or more distal pocket mutations.In some embodiments, the H-NOX protein or H-NOX domain of a polymericH-NOX protein comprises one, two, three, four, five, six, seven, eight,nine, ten or more than ten distal pocket mutations. In some embodiments,the distal pocket mutation corresponds to a L144F mutation of T.tengcongensis H-NOX. In some embodiments, the distal pocket mutation isa L144F mutation of T. tengcongensis H-NOX. In some embodiments, H-NOXprotein or the H-NOX domain of a polymeric H-NOX protein comprises twodistal pocket mutations. In some embodiments, the H-NOX protein or H-NOXdomain of a polymeric H-NOX protein corresponds to a W9F/L144F mutationof T. tengcongensis H-NOX. In some embodiments, the H-NOX protein orH-NOX domain of a polymeric H-NOX protein is a W9F/L144F mutation of T.tengcongensis H-NOX.

Residues that are not in the distal pocket can also affect thethree-dimensional structure of the heme group; this structure in turnaffects the binding of O₂ and NO to iron in the heme group. Accordingly,in some embodiments, the H-NOX protein or H-NOX domain of a polymericH-NOX protein has one or more mutations outside of the distal pocket.Examples of residues that can be mutated but are not in the distalpocket include Pro115 and Arg135 of T. tengcongensis H-NOX. In someembodiments, the mutation is in the proximal pocket which includesHis105 as a residue that ligates to the heme iron.

In some embodiments when two or more mutations are present; at least onemutation is in the distal pocket, and at least one mutation is outsideof the distal pocket (e.g., a mutation in the proximal pocket). In someembodiments, all the mutations are in the distal pocket.

To reduce the immunogenicity of H-NOX protein or H-NOX domains derivedfrom sources other than humans, amino acids in an H-NOX protein or H-NOXdomain can be mutated to the corresponding amino acids in a human H-NOX.For example, one or more amino acids on the surface of the tertiarystructure of a non-human H-NOX protein or H-NOX domain can be mutated tothe corresponding amino acid in a human H-NOX protein or H-NOX domain.In some variations, mutation of one or more surface amino acids may becombined with mutation of two or more distal pocket residues, mutationof one or more residues outside of the distal pocket (e.g., a mutationin the proximal pocket), or combinations of two or more of theforegoing.

The invention also relates to any combination of mutation describedherein, such as double, triple, or higher multiple mutations. Forexample, combinations of any of the mutations described herein can bemade in the same H-NOX protein. Note that mutations in equivalentpositions in other mammalian or non-mammalian H-NOX proteins are alsoencompassed by this invention. Exemplary mutant H-NOX proteins or mutantH-NOX domains comprise one or more mutations that impart altered O₂ orNO ligand-binding relative to the corresponding wild-type H-NOX domainand are operative as a physiologically compatible mammalian O₂ blood gascarrier.

The residue number for a mutation indicates the position in the sequenceof the particular H-NOX protein being described. For example, T.tengcongensis I5A refers to the replacement of isoleucine by alanine atthe fifth position in T. tengcongensis H-NOX. The same isoleucine toalanine mutation can be made in the corresponding residue in any otherH-NOX protein or H-NOX domain (this residue may or may not be the fifthresidue in the sequence of other H-NOX proteins). Since the amino acidsequences of mammalian β1 H-NOX domains differ by at most two aminoacids, mutations that produce desirable mutant H-NOX proteins or H-NOXdomains when introduced into wild-type rat β1 H-NOX proteins are alsoexpected to produce desirable mutant H-NOX proteins or H-NOX domainswhen introduced into wild-type β1 H-NOX proteins or H-NOX domains fromother mammals, such as humans.

In some embodiments, the H-NOX protein is a trimer comprising three T.tengcongensis L144F H-NOX domains and three foldon domains. In someembodiments, the H-NOX protein is a trimer comprising three T.tengcongensis W9F/L144F H-NOX domains and three foldon domains. In someembodiments, the H-NOX protein is a trimer comprising three T.tengcongensis wildtype H-NOX domains and three foldon domains.

Modifications to H-NOX Proteins

Any of the wild-type or mutant H-NOX proteins, including polymeric H-NOXproteins, can be modified and/or formulated using standard methods toenhance therapeutic or industrial applications. For example, andparticularly as applied to heterologous engineered H-NOX proteins, avariety of methods are known in the art for insulating such agents fromimmune surveillance, including crosslinking, PEGylation, carbohydratedecoration, etc. (e.g., Rohlfs, R. J. et al. (May 15, 1998). J. Biol.Chem. 273(20):12128-12134; Migita, R. et al. (June 1997). J. Appl.Physiol. 82(6):1995-2002; Vandegriff, K. D. et al. (Aug. 15, 2004).Biochem J. 382(Pt 1):183-189, which are each hereby incorporated byreference in their entireties, particularly with respect to themodification of proteins) as well as other techniques known to theskilled artisan. Fusing an H-NOX protein, including a polymeric H-NOXprotein, with a human protein such as human serum albumin can increasethe serum half-life, viscosity, and colloidal oncotic pressure. In someembodiments, an H-NOX protein is modified during or after its synthesisto decrease its immunogenicity and/or to increase its plasma retentiontime. H-NOX proteins can also be encapsulated (such as encapsulationwithin liposomes or nanoparticles).

In some embodiments, the H-NOX protein comprises one of more tags; e.g.to assist in purification of the H-NOX protein. Examples of tagsinclude, but are not limited to His₆, FLAG, GST, and MBP. In someembodiments, the H-NOX protein comprises one of more His₆ tags. The oneor more His₆ tags may be removed prior to use of the polymeric H-NOXprotein; e.g. by treatment with an exopeptidase. In some embodiments,the H-NOX protein is a trimer comprising three T. tengcongensis L144FH-NOX domains, three foldon domains, and three His₆ tags. In someembodiments, the H-NOX protein is a trimer comprising three T.tengcongensis W9F/L144F H-NOX domains, three foldon domains, and threeHis₆ tags. In some embodiments, the H-NOX protein is a trimer comprisingthree T. tengcongensis wildtype H-NOX domains, three foldon domains, andthree His₆ tags.

In some embodiments, the H-NOX protein comprises one or morepolyethylene glycol (PEG) molecules (i.e., is PEGylated). In someembodiments, the H-NOX protein is a trimer comprising three T.tengcongensis L144F H-NOX domains, three foldon domains, and one or morepolyethylene glycol molecules (PEGylated trimer Tt H-NOX L144F). In someembodiments, the H-NOX protein is a trimer comprising three T.tengcongensis W9F/L144F H-NOX domains, three foldon domains, and one ormore polyethylene glycol molecules. In some embodiments, the H-NOXprotein is a trimer comprising three T. tengcongensis wildtype H-NOXdomains, three foldon domains, and one or more polyethylene glycolmolecules. In some embodiments, the molecular weight of the PEG isbetween about 1 kDa and about 50 kDa. In some embodiments, the molecularweight of the PED is between about any of 1 kDa and 50 kDa, 1 kDa and 40kDa, 1 kDa and 30 kDa, 1 kDa and 25 kDa, 1 kDa and 20 kDa, 1 kDa and 15kDa, 1 kDa and 10 kDa, 1 kDa and 5 kDa, 5 kDa and 50 kDa, 5 kDa and 40kDa, 5 kDa and 30 kDa, 5 kDa and 25 kDa, 5 kDa and 20 kDa, 5 kDa and 15kDa, 5 kDa and 10 kDa, 10 kDa and 50 kDa, 10 kDa and 40 kDa, 10 kDa and30 kDa, 10 kDa and 25 kDa, 10 kDa and 20 kDa, 10 kDa and 15 kDa, 15 kDaand 50 kDa, 15 kDa and 40 kDa, 15 kDa and 35 kDa, 15 kDa and 30 kDa, 15kDa and 25 kDa, 15 kDa and 20 kDa, 20 kDa and 50 kDa, 20 kDa and 40 kDa,20 kDa and 30 kDa, 20 kDa and 25 kDa, 25 kDa and 50 kDa, 25 kDa and 40kDa, 25 kDa and 30 kDa, 30 kDa and 50 kDa, 30 kDa and 40 kDa, or 40 kDaand 50 kDa. In some embodiments, the H-NOX protein comprises any one ofmore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, or 50 PEG molecules per H-NOX monomer or any number therebetween. Insome embodiments, the H-NOX protein comprises an average of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 PEG molecules perH-NOX monomer or any number therebetween.

Polymerization Domains

In some aspects, the invention provides polymeric H-NOX proteinscomprising two or more H-NOX domains and one or more polymerizationdomains. Polymerization domains are used to link two or more H-NOXdomains to form a polymeric H-NOX protein. One or more polymerizationdomains may be used to produce dimers, trimers, tetramers, pentamers,etc. of H-NOX proteins. Polymerization domains are known in the art,such as: the foldon of T4 bacteriophage fibritin, Arc, POZ, coiled coildomains (including GCN4, leucine zippers, Velcro), uteroglobin,collagen, 3-stranded coiled colis (matrilin-1), thrombosporins, TRPV1-C,P53, Mnt, avadin, streptavidin, Bcr-Abl, COMP, verotoxin subunit B,CamKII, RCK, and domains from N ethylmaleimide-sensitive fusion protein,STM3548, KaiC, TyrR, Hcp1, CcmK4, GP41, anthrax protective antigen,aerolysin, a-hemolysin, C4b-binding protein, Mi-CK, arylsurfatase A, andviral capsid proteins. The polymerization domains may be covalently ornon-covalently linked to the H-NOX domains. In some embodiments, apolymerization domain is linked to an H-NOX domain to form a monomersubunit such that the polymerization domains from a plurality of monomersubunits associate to form a polymeric H-NOX domain. In someembodiments, the C-terminus of an H-NOX domain is linked to theN-terminus of a polymerization domain. In other embodiments, theN-terminus of an H-NOX domain is linked to the N-terminus of apolymerization domain. In yet other embodiments, the C-terminus of anH-NOX domain is linked to the C-terminus of a polymerization domain. Insome embodiments, the N-terminus of an H-NOX domain is linked to theC-terminus of a polymerization domain.

Linkers may be used to join a polymerization domain to an H-NOX domain;for example, for example, amino acid linkers. In some embodiments, alinker comprising any one of one, two, three, four, five, six, seven,eight, nine, ten or more than ten amino acids may be placed between thepolymerization domain and the H-NOX domain. Exemplary linkers includebut are not limited to Gly-Ser-Gly and Arg-Gly-Ser linkers.

Bacteriophage T4 Fibritin Trimerization Domain

An exemplary polymerization domain is the foldon domain of bacteriophageT4. The wac gene from the bacteriophage T4 encodes the fibritin protein,a 486 amino acid protein with a C-terminal trimerization domain(residues 457-483) (Efimov, V. P. et al. (1994) J Mol Biol 242:470-486).The domain is able to trimerize fibritin both in vitro and in vivo(Boudko, S. P. et al. (2002) Eur J Biochem 269:833-841; Letarov, A. V.,et al., (1999) Biochemistry (Mosc) 64:817-823; Tao, Y., et al., (1997)Structure 5:789-798). The isolated 27 residue trimerization domain,often referred to as the “foldon domain,” has been used to constructchimeric trimers in a number of different proteins (including HIVenvelope glycoproteins (Yang, X. et al., (2002) J Virol 76:4634-4642),adenoviral adhesins (Papanikolopoulou, K., et al., (2004) J Biol Chem279:8991-8998; Papanikolopoulou, K. et al. (2004) J Mol Biol342:219-227), collagen (Zhang, C., et al. (2009) Biotechnol Prog25:1660-1668), phage P22 gp26 (Bhardwaj, A., et al. (2008) Protein Sci17:1475-1485), and rabies virus glycoprotein (Sissoeff, L., et al.(2005) J Gen Virol 86:2543-2552). An exemplary sequence of the foldondomain is shown in FIG. 1 and provided by SEQ ID NO:4.

The isolated foldon domain folds into a single 0-hairpin structure andtrimerizes into a β-propeller structure involving three hairpins (Guthe,S. et al. (2004) J Mol Biol 337:905-915). The structure of the foldondomain alone has been determined by NMR (Guthe, S. et al. (2004) J MolBiol 337:905-915) and the structures of several proteins trimerized withthe foldon domain have been solved by X-ray crystallography(Papanikolopoulou, K., et al., (2004) J Biol Chem 279:8991-8998;Stetefeld, J. et al. (2003) Structure 11:339-346; Yokoi, N. et al.(2010) Small 6:1873-1879). The domain folds and trimerizes rapidlyreducing the opportunity for misfolding intermediates or off-pathwayoligomerization products (Guthe, S. et al. (2004) J Mol Biol337:905-915). The foldon domain is very stable, able to maintaintertiary structure and oligomerization in >10% SDS, 6.0 M guanidinehydrochloride, or 80° C. (Bhardwaj, A., et al. (2008) Protein Sci17:1475-1485; Bhardwaj, A., et al. (2007) J Mol Biol 371:374-387) andcan improve the stability of sequences fused to the foldon domain (Du,C. et al. (2008) Appl Microbiol Biotechnol 79:195-202.

In some embodiments, the C-terminus of an H-NOX domain is linked to theN-terminus of a foldon domain. In other embodiments, the N-terminus ofan H-NOX domain is linked to the N-terminus of a foldon domain. In yetother embodiments, the C-terminus of an H-NOX domain is linked to theC-terminus of a foldon domain. In some embodiments, the N-terminus of anH-NOX domain is linked to the C-terminus of a foldon domain.

In some embodiments, linkers are be used to join a foldon domain to anH-NOX domain. In some embodiments, a linker comprising any one of one,two, three, four, five, six, seven, eight, nine, ten or more than tenamino acids may be placed between the polymerization domain and theH-NOX domain. Exemplary linkers include but are not limited toGly-Ser-Gly and Arg-Gly-Ser linkers. In some embodiments, the inventionprovides a trimeric H-NOX protein comprising from N-terminus toC-terminus: a T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acidlinker, and a foldon domain. In some embodiments, the invention providesa trimeric H-NOX protein comprising from N-terminus to C-terminus: a T.tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker, a foldondomain, an Arg-Gly-Ser amino acid linker, and a His₆ tag. In someembodiments, the T. tengcongensis H-NOX domain comprises an L144Fmutation. In some embodiments, the T. tengcongensis H-NOX domaincomprises a W9F mutation and a L144F mutation. In some embodiments, theT. tengcongensis H-NOX domain is a wild-type H-NOX domain.

Monomeric H-NOX Domain Subunits

In one aspect, the invention provides recombinant monomeric H-NOXproteins (i.e. monomeric H-NOX subunits of polymeric H-NOX proteins)that can associate to form polymeric H-NOX proteins. In someembodiments, the invention provides recombinant H-NOX proteinscomprising an H-NOX domain as described herein and a polymerizationdomain. The H-NOX domain and the polymerization domain may be covalentlylinked or noncovalently linked. In some embodiments, the C-terminus ofan H-NOX domain of the recombinant monomeric H-NOX protein is linked tothe N-terminus of a polymerization domain. In other embodiments, theN-terminus of an H-NOX domain of the recombinant monomeric H-NOX proteinis linked to the N-terminus of a polymerization domain. In yet otherembodiments, the C-terminus of an H-NOX domain of the recombinantmonomeric H-NOX protein is linked to the C-terminus of a polymerizationdomain. In some embodiments, the N-terminus of an H-NOX domain of therecombinant monomeric H-NOX protein is linked to the C-terminus of apolymerization domain. In some embodiments, the recombinant monomericH-NOX protein does not comprise a guanylyl cyclase domain.

In some embodiments, the monomeric H-NOX protein comprises a wild-typeH-NOX domain. In some embodiments of the invention, the monomeric H-NOXprotein comprises one of more mutations in the H-NOX domain. In someembodiments, the one or more mutations alter the O₂ dissociationconstant, the k_(off) for oxygen, the rate of heme autooxidation, the NOreactivity, the NO stability or any combination of two or more of theforegoing compared to that of the corresponding wild-type H-NOX domain.In some embodiments, the mutation is a distal pocket mutation. In someembodiments, the mutation comprises a mutation that is not in the distalpocket. In some embodiments, the distal pocket mutation corresponds to aL144 mutation of T. tengcongensis (e.g. a L144F mutation). In someembodiments, the recombinant monomeric H-NOX protein comprises twodistal pocket mutations corresponding to a W9 and a L144 mutation of T.tengcongensis (e.g. a W9F/L144F mutation).

In some aspects, the invention provides recombinant monomeric H-NOXproteins that associate to form trimeric H-NOX proteins. In someembodiments, the recombinant H-NOX protein comprises an H-NOX domain anda trimerization domain. In some embodiments, the trimerization domain isa foldon domain as discussed herein. In some embodiments, the H-NOXdomain is a T. tengcongensis H-NOX domain. In some embodiments theC-terminus of the T. tengcongensis H-NOX domain is covalently linked tothe N-terminus of the foldon domain. In some embodiments the C-terminusof the T. tengcongensis H-NOX domain is covalently linked to theC-terminus of the foldon domain. In some embodiments, the T.tengcongensis domain is an L144F H-NOX domain. In some embodiments, theT. tengcongensis domain is a W9F/L144F H-NOX domain. In someembodiments, the T. tengcongensis domain is a wild-type H-NOX domain.

In some embodiments, the H-NOX domain is covalently linked to thepolymerization domain using an amino acid linker sequence. In someembodiments, the amino acid linker sequence is one, two, three, four,five, six, seven, eight, nine, ten or more than ten amino acids inlength. Exemplary amino acid linker sequences include but are notlimited to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence. In someembodiments, the polymeric H-NOX protein is a trimeric H-NOX proteincomprising three H-NOX domains and three trimerization sequences whereinthe H-NOX domain is covalently linked to the trimerization domain via anamino acid linker sequence. In some embodiments, the monomeric H-NOXprotein comprises the following from the N-terminus to the C-terminus:an L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linkersequence, and a foldon domain. In some embodiments, the monomeric H-NOXprotein comprises the following from the N-terminus to the C-terminus: aW9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linkersequence, and a foldon domain. In some embodiments, the monomeric H-NOXprotein comprises the following from the N-terminus to the C-terminus: awild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linkersequence, and a foldon domain.

In some embodiments, the recombinant monomeric H-NOX protein comprises atag; e.g., a His₆, a FLAG, a GST, or an MBP tag. In some embodiments,the recombinant monomeric H-NOX protein comprises a His₆ tag. In someembodiments, the recombinant monomeric H-NOX protein does not comprise atag. In some embodiments, the tag (e.g. a His₆ tag) is covalently linkedto the polymerization domain using an amino acid spacer sequence. Insome embodiments, the amino acid linker sequence is one, two, three,four, five, six, seven, eight, nine, ten or more than ten amino acids inlength. Exemplary amino acid linker sequences include but are notlimited to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence. In someembodiments, the polymeric H-NOX protein is a trimeric H-NOX proteincomprising three H-NOX domains, three trimerization sequences, and threeHis₆ tags, wherein the H-NOX domain is covalently linked to thetrimerization domain via an amino acid linker sequence and thetrimerization domain is covalently linked to the His₆ tag via an aminoacid linker sequence. In some embodiments, the monomeric H-NOX proteincomprises the following from the N-terminus to the C-terminus: an L144FT. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence,a foldon domain, an Arg-Gly-Ser linker sequence, and a His₆ tag. In someembodiments, the monomeric H-NOX protein comprises the following fromthe N-terminus to the C-terminus: a W9F/L144F T. tengcongensis H-NOXdomain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, anArg-Gly-Ser linker sequence, and a His₆ tag. In some embodiments, themonomeric H-NOX protein comprises the following from the N-terminus tothe C-terminus: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Glyamino acid linker sequence, a foldon domain, an Arg-Gly-Ser linkersequence, and a His₆ tag.

In some embodiments the recombinant monomeric H-NOX protein comprisesthe amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8.

Characteristics of Wild-Type and Mutant H-NOX Proteins

The present invention provides the use of O₂ carrier polypeptides foruse in enhancing tumor immunogenicity; for example, by inhibiting theimmune suppressive activities associated with tumor hypoxia. Anon-limiting exemplary family of O₂ carrier polypeptides is the H-NOXfamily of O₂ carrier polypeptides. As described herein, a large numberof diverse H-NOX mutant proteins, including polymeric H-NOX proteins,providing ranges of NO and O₂ dissociation constants, O₂ k_(off), NOreactivity, and stability have been generated. To provide operativeblood gas carriers, the H-NOX proteins may be used to functionallyreplace or supplement endogenous O₂ carriers, such as hemoglobin. Insome embodiments, H-NOX proteins such as polymeric H-NOX proteins, areused to deliver O₂ to hypoxic tumor tissue (e.g. a glioblastoma) as anadjuvant to radiation therapy or chemotherapy. Accordingly, in someembodiments, an H-NOX protein has a similar or improved O₂ associationrate, O₂ dissociation rate, dissociation constant for O₂ binding, NOstability, NO reactivity, autoxidation rate, plasma retention time, orany combination of two or more of the foregoing compared to anendogenous O₂ carrier, such as hemoglobin. In some embodiments, theH-NOX protein is a polymeric H-NOX protein. In some embodiments, thepolymeric H-NOX protein is a trimeric H-NOX protein comprising threemonomers, each monomer comprising a T. tengcongensis L144F H-NOX domainand a foldon domain. In some embodiments, the polymeric H-NOX protein isa trimeric H-NOX protein comprising three monomers, each monomercomprising a T. tengcongensis W9F/L144F H-NOX domain and a foldondomain. In some embodiments, the polymeric H-NOX protein is a trimericH-NOX protein comprising three monomers, each monomer comprising a T.tengcongensis L144F H-NOX domain and a foldon domain.

In various embodiments, the k_(off) for O₂ for an H-NOX protein,including a polymeric H-NOX protein, is between about 0.01 to about 200s⁻¹ at 20° C., such as about 0.1 to about 200 s⁻¹, about 0.1 to 100 s⁻,about 1.0 to about 16.0 s⁻¹, about 1.35 to about 23.4 s⁻, about 1.34 toabout 18 s⁻¹, about 1.35 to about 14.5 s⁻¹, about 0.21 to about 23.4s⁻¹, about 1.35 to about 2.9 s⁻¹ about 2 to about 3 s⁻¹, about 5 toabout 15 s⁻¹, or about 0.1 to about 1 s⁻¹. In some embodiments, theH-NOX protein has a k_(off) for oxygen that is less than or equal toabout 0.65 s⁻¹ at 20° C. (such as between about 0.21 s⁻¹ to about 0.65s⁻¹ at 20° C.).

In various embodiments, the k_(on) for O₂ for an H-NOX protein,including a polymeric H-NOX protein, is between about 0.14 to about 60μM⁻¹s⁻¹ at 20° C., such as about 6 to about 60 μM⁻¹s⁻¹, about 6 to 12μM⁻¹s⁻¹, about 15 to about 60 μM⁻¹s⁻¹, about 5 to about 18 μM⁻¹s⁻¹, orabout 6 to about 15 μM⁻¹s⁻¹.

In various embodiments, the kinetic or calculated K_(D) for O₂ bindingby an H-NOX protein, including a polymeric H-NOX protein, is betweenabout 1 nM to 1 mM, about 1 μM to about 10 μM, or about 10 μM to about50 μM. In some embodiments the calculated K_(D) for O₂ binding is anyone of about 2 nM to about 2 μM, about 2p M to about 1 mM, about 100 nMto about 1 μM, about 9 μM to about 50 μM, about 100 μM to about 1 mM,about 50 nM to about 10 μM, about 2 nM to about 50 μM, about 100 nM toabout 1.9 μM, about 150 nM to about 1 μM, or about 100 nM to about 255nM, about 20 nM to about 2 μM, 20 nM to about 75 nM, about 1 μM to about2 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, or about 100nM to 500 nM at 20° C. In some embodiments, the kinetic or calculatedK_(D) for O₂ binding is less than about any of 100 nM, 80 nM, 50 nM, 30nM, 25 nM, 20 nM, or 10 nM at 20° C.

In various embodiments, the kinetic or calculated K_(D) for O₂ bindingby an H-NOX protein, including a polymeric H-NOX protein, is withinabout 0.01 to about 100-fold of that of hemoglobin under the sameconditions (such as at 20° C.), such as between about 0.1 to about10-fold or between about 0.5 to about 2-fold of that of hemoglobin underthe same conditions (such as at 20° C.). In various embodiments, thekinetic or calculated K_(D) for NO binding by an H-NOX protein is withinabout 0.01 to about 100-fold of that of hemoglobin under the sameconditions (such as at 20° C.), such as between about 0.1 to about10-fold or between about 0.5 to about 2-fold of that of hemoglobin underthe same conditions (such as at 20° C.).

In some embodiments, less than about any of 50, 40, 30, 10, or 5% of anH-NOX protein, including a polymeric H-NOX protein, is oxidized afterincubation for about any of 1, 2, 4, 6, 8, 10, 15, or 20 hours at 20° C.

In various embodiments, the NO reactivity of an H-NOX protein, includinga polymeric H-NOX protein, is less than about 700 s⁻¹ at 20° C., such asless than about 600 s⁻¹, 500 s⁻¹, 400 s⁻¹, 300 s⁻¹, 200 s⁻¹, 100 s⁻¹, 75s⁻¹, 50 s⁻¹, 25 s⁻¹, 20 s⁻¹, 10 s⁻¹, 50 s⁻¹, 3 s⁻¹, 2 s⁻¹, 1.8 s⁻¹, 1.5s⁻¹, 1.2 s⁻¹, 1.0 s⁻¹, 0.8 s⁻¹, 0.7 s⁻¹, or 0.6 s⁻¹ at 20° C. In variousembodiments, the NO reactivity of an H-NOX protein is between about 0.1to about 600 s⁻¹ at 20° C., such as between about 0.5 to about 400 s⁻¹,about 0.5 to about 100 s⁻¹, about 0.5 to about 50 s⁻¹, about 0.5 toabout 10 s⁻¹, about 1 to about 5 s⁻¹, or about 0.5 to about 2.1 s⁻¹ at20° C. In various embodiments, the reactivity of an H-NOX protein is atleast about 10, 100, 1,000, or 10,000 fold lower than that of hemoglobinunder the same conditions, such as at 20° C.

In various embodiments, the rate of heme autoxidation of an H-NOXprotein, including a polymeric H-NOX protein, is less than about 1.0h-tat 37° C., such as less than about any of 0.9 h⁻¹, 0.8 h⁻¹, 0.7 h⁻¹,0.6 h⁻¹, 0.5 h⁻¹, 0.4 h⁻¹, 0.3 h⁻¹, 0.2 h⁻¹, 0.1 h⁻¹, or 0.05 h⁻¹ at 37C. In various embodiments, the rate of heme autoxidation of an H-NOXprotein is between about 0.006 to about 5.0 h⁻¹ at 37° C., such as about0.006 to about 1.0 h⁻¹, 0.006 to about 0.9 h⁻¹, or about 0.06 to about0.5 h⁻¹ at 37° C.

In various embodiments, a mutant H-NOX protein, including a polymericH-NOX protein, has (a) an O₂ or NO dissociation constant, associationrate (k_(on) for O₂ or NO), or dissociation rate (k_(off) for O₂ or NO)within 2 orders of magnitude of that of hemoglobin, (b) has an NOaffinity weaker (e.g., at least about 10-fold, 100-fold, or 1000-foldweaker) than that of sGC β1, respectively, (c) an NO reactivity withbound 02 at least 1000-fold less than hemoglobin, (d) an in vivo plasmaretention time at least 2, 10, 100, or 1000-fold higher than that ofhemoglobin, or (e) any combination of two or more of the foregoing.

Exemplary suitable O₂ carriers provide dissociation constants within twoorders of magnitude of that of hemoglobin, i.e. between about 0.01 and100-fold, such as between about 0.1 and 10-fold, or between about 0.5and 2-fold of that of hemoglobin. A variety of established techniquesmay be used to quantify dissociation constants, such as the techniquesdescribed herein (Boon, E. M. et al. (2005). Nature Chem. Biol. 1:53-59;Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446;Boon. E. M. et al. (2005). J. Inorg. Biochem. 99(4):892-902),Vandegriff, K. D. et al. (Aug. 15, 2004). Biochem J. 382(Pt 1):183-189,which are each hereby incorporated by reference in their entireties,particularly with respect to the measurement of dissociation constants),as well as those known to the skilled artisan. Exemplary O₂ carriersprovide low or minimized NO reactivity of the H-NOX protein with boundO₂, such as an NO reactivity lower than that of hemoglobin. In someembodiments, the NO reactivity is much lower, such as at least about 10,100, 1,000, or 10,000-fold lower than that of hemoglobin. A variety ofestablished techniques may be used to quantify NO reactivity (Boon, E.M. et al. (2005). Nature Chem. Biol. 1:53-59; Boon, E. M. et al.(October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al.(2005). J. Inorg. Biochem. 99(4):892-902), Vandegriff, K. D. et al.(Aug. 15, 2004). Biochem J. 382(Pt 1):183-189, which are each herebyincorporated by reference in their entireties, particularly with respectto the measurement of NO reactivity) as well as those known to theskilled artisan. Because wild-type T. tengcongensis H-NOX has such a lowNO reactivity, other wild-type H-NOX proteins and mutant H-NOX proteinsmay have a similar low NO reactivity. For example, T. tengcongensisH-NOX Y140H has an NO reactivity similar to that of wild-type T.tengcongensis H-NOX.

In addition, suitable O₂ carriers provide high or maximized stability,particularly in vivo stability. A variety of stability metrics may beused, such as oxidative stability (e.g., stability to autoxidation oroxidation by NO), temperature stability, and in vivo stability. Avariety of established techniques may be used to quantify stability,such as the techniques described herein (Boon, E. M. et al. (2005).Nature Chem. Biol. 1:53-59; Boon, E. M. et al. (October 2005). Curr.Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg.Biochem. 99(4):892-902), as well as those known to the skilled artisan.For in vivo stability in plasma, blood, or tissue, exemplary metrics ofstability include retention time, rate of clearance, and half-life.H-NOX proteins from thermophilic organisms are expected to be stable athigh temperatures. In various embodiments, the plasma retention timesare at least about 2-, 10-, 100-, or 1000-fold greater than that ofhemoglobin (e.g. Bobofchak, K. M. et al. (August 2003). Am. J. Physiol.Heart Circ. Physiol. 285(2):H549-H561). As will be appreciated by theskilled artisan, hemoglobin-based blood substitutes are limited by therapid clearance of cell-free hemoglobin from plasma due the presence ofreceptors for hemoglobin that remove cell-free hemoglobin from plasma.Since there are no receptors for H-NOX proteins in plasma, wild-type andmutant H-NOX proteins are expected to have a longer plasma retentiontime than that of hemoglobin. If desired, the plasma retention time canbe increased by PEGylating or crosslinking an H-NOX protein or fusing anH-NOX protein with another protein using standard methods (such as thosedescribed herein and those known to the skilled artisan).

In various embodiments, the H-NOX protein, including a polymeric H-NOXprotein, has an O₂ dissociation constant between about 1 nM to about 1mM at 20° C. and a NO reactivity at least about 10-fold lower than thatof hemoglobin under the same conditions, such as at 20° C. In someembodiments, the H-NOX protein has an O₂ dissociation constant betweenabout 1 nM to about 1 mM at 20° C. and a NO reactivity less than about700 s⁻¹ at 20° C. (e.g., less than about 600 s⁻¹, 500 s⁻¹, 100 s⁻¹, 20s⁻¹, or 1.8 s⁻¹ at 20° C.). In some embodiments, the H-NOX protein hasan O₂ dissociation constant within 2 orders of magnitude of that ofhemoglobin and a NO reactivity at least about 10-fold lower than that ofhemoglobin under the same conditions, such as at 20° C. In someembodiments, the H-NOX protein has a k_(off) for oxygen between about0.01 to about 200 s⁻¹ at 20° C. and an NO reactivity at least about10-fold lower than that of hemoglobin under the same conditions, such asat 20° C. In some embodiments, the H-NOX protein has a k_(off) foroxygen that is less than about 0.65 s⁻¹ at 20° C. (such as between about0.21 s⁻¹ to about 0.64 s⁻¹ at 20° C.) and a NO reactivity at least about10-fold lower than that of hemoglobin under the same conditions, such asat 20° C. In some embodiments of the invention, the O₂ dissociationconstant of the H-NOX protein is between about 1 nM to about 1 μM (1000nM), about 1 μM to about 10 μM, or about 10 μM to about 50 μM. Inparticular embodiments, the O₂ dissociation constant of the H-NOXprotein is between about 2 nM to about 50 μM, about 50 nM to about 10μM, about 100 nM to about 1.9 μM, about 150 nM to about 1 μM, or about100 nM to about 255 nM at 20° C. In various embodiments, the O₂dissociation constant of the H-NOX protein is less than about 80 nM at20° C., such as between about 20 nM to about 75 nM at 20° C. In someembodiments, the NO reactivity of the H-NOX protein is at least about100-fold lower or about 1,000 fold lower than that of hemoglobin, underthe same conditions, such as at 20° C. In some embodiments, the NOreactivity of the H-NOX protein is less than about 700 s⁻¹ at 20° C.,such as less than about 600 s⁻¹, 500 s⁻¹, 400 s⁻¹, 300 s⁻¹, 200 s⁻¹, 100s⁻¹, 75 s⁻¹, 50 s⁻¹, 25 s⁻¹, 20 s⁻¹, 10 s⁻¹, 50 s⁻¹, 3 s⁻¹, 2 s⁻¹, 1.8s⁻¹, 1.5 s⁻¹, 1.2 s⁻¹, 1.0 s⁻¹, 0.8 s⁻¹, 0.7 s⁻¹, or 0.6 s⁻¹ at 20° C.In some embodiments, the k_(off) for oxygen of the H-NOX protein isbetween 0.01 to 200 s⁻¹ at 20° C., such as about 0.1 to about 200 s⁻¹,about 0.1 to 100 s⁻¹, about 1.35 to about 23.4 s⁻¹, about 1.34 to about18 s⁻¹, about 1.35 to about 14.5 s⁻¹, about 0.21 to about 23.4 s⁻¹,about 2 to about 3 s⁻¹, about 5 to about 15 s⁻¹, or about 0.1 to about 1s⁻¹. In some embodiments, the O₂ dissociation constant of the H-NOXprotein is between about 100 nM to about 1.9 μM at 20° C., and thek_(off) for oxygen of the H-NOX protein is between about 1.35 s⁻¹ toabout 14.5 s⁻¹ at 20° C. In some embodiments, the rate of hemeautoxidation of the H-NOX protein is less than about 1 h⁻¹ at 37° C.,such as less than about any of 0.9 h⁻¹, 0.8 h⁻¹, 0.7 h⁻¹, 0.6 h⁻¹, 0.5h⁻¹, 0.4 h⁻¹, 0.3 h⁻¹, 0.2 h⁻¹, or 0.1 h⁻¹. In some embodiments, thek_(off) for oxygen of the H-NOX protein is between about 1.35 s⁻¹ toabout 14.5 s⁻¹ at 20° C., and the rate of heme autoxidation of the H-NOXprotein is less than about 1 h⁻¹ at 37° C. In some embodiments, thek_(off) for oxygen of the H-NOX protein is between about 1.35 s⁻¹ toabout 14.5 s⁻¹ at 20° C., and the NO reactivity of the H-NOX protein isless than about 700 s⁻¹ at 20° C. (e.g., less than about 600 s⁻¹, 500s⁻¹, 100 s⁻¹, 20 s⁻¹, or 1.8 s⁻¹ at 20° C.). In some embodiments, therate of heme autoxidation of the H-NOX protein is less than about 1 h⁻¹at 37° C., and the NO reactivity of the H-NOX protein is less than about700 s⁻¹ at 20° C. (e.g., less than about 600 s⁻¹, 500 s⁻¹, 100 s⁻¹, 20s⁻¹, or 1.8 s⁻¹ at 20° C.).

In some embodiments, the viscosity of the H-NOX protein solution,including a polymeric H-NOX protein solution, is between 1 and 4centipoise (cP). In some embodiments, the colloid oncotic pressure ofthe H-NOX protein solution is between 20 and 50 mm Hg.

Measurement of O₂ and/or NO Binding

One skilled in the art can readily determine the oxygen and nitric oxidebinding characteristics of any H-NOX protein including a polymeric H-NOXprotein such as a trimeric H-NOX protein by methods known in the art andby the non-limiting exemplary methods described below.

Kinetic K_(D): Ratio of k_(off) to k_(on)

The kinetic K_(D) value is determined for wild-type and mutant H-NOXproteins, including polymeric H-NOS proteins, essentially as describedby Boon, E. M. et al. (2005). Nature Chemical Biology 1:53-59, which ishereby incorporated by reference in its entirety, particularly withrespect to the measurement of O₂ association rates, O₂ dissociationrates, dissociation constants for O₂ binding, autoxidation rates, and NOdissociation rates.

k_(on) (O₂ Association Rate)

O₂ association to the heme is measured using flash photolysis at 20° C.It is not possible to flash off the Fe^(II)-O₂ complex as a result ofthe very fast geminate recombination kinetics; thus, the Fen-CO complexis subjected to flash photolysis with laser light at 560 nm(Hewlett-Packard, Palo Alto, Calif.), producing the 5-coordinate Fe^(II)intermediate, to which the binding of molecular O₂ is followed atvarious wavelengths. Protein samples are made by anaerobic reductionwith 10 mM dithionite, followed by desalting on a PD-10 column(Millipore, Inc., Billerica, Mass.). The samples are then diluted to 20μM heme in 50 mM TEA, 50 mM NaCl, pH 7.5 buffer in acontrolled-atmosphere quartz cuvette, with a size of 100 μL to 1 mL anda path-length of 1-cm. CO gas is flowed over the headspace of thiscuvette for 10 minutes to form the Fe^(II)-CO complex, the formation ofwhich is verified by UV-visible spectroscopy (Soret maximum 423 nm).This sample is then either used to measure CO-rebinding kinetics afterflash photolysis while still under 1 atmosphere of CO gas, or it isopened and stirred in air for 30 minutes to fully oxygenate the bufferbefore flash photolysis to watch O₂-rebinding events. O₂ association tothe heme is monitored at multiple wavelengths versus time. These tracesare fit with a single exponential using Igor Pro software (Wavemetrics,Inc., Oswego, Oreg.; latest 2005 version). This rate is independent ofobservation wavelength but dependent on O₂ concentration. UV-visiblespectroscopy is used throughout to confirm all the complexes andintermediates (Cary 3K, Varian, Inc. Palo Alto, Calif.). Transientabsorption data are collected using instruments described in Dmochowski,I. J. et al. (Aug. 31, 2000). J Inorg Biochem. 81(3):221-228, which ishereby incorporated by reference in its entirety, particularly withrespect to instrumentation. The instrument has a response time of 20 ns,and the data are digitized at 200 megasamples s⁻¹.

k_(off) (O₂ Dissociation Rate)

To measure the k_(off), Fe^(II)-O₂ complexes of protein (5 μM heme), arediluted in anaerobic 50 mM TEA, 50 mM NaCl, pH 7.5 buffer, and arerapidly mixed with an equal volume of the same buffer (anaerobic)containing various concentrations of dithionite and/or saturating COgas. Data are acquired on a HI-TECH Scientific SF-61 stopped-flowspectrophotometer equipped with a Neslab RTE-100 constant-temperaturebath set to 20° C. (TGK Scientific LTD., Bradford On Avon, UnitedKingdom). The dissociation of O₂ from the heme is monitored as anincrease in the absorbance at 437 nm, a maximum in theFe^(II)—Fe^(II)—O₂ difference spectrum, or 425 nm, a maximum in theFe^(II)—Fe^(II)—CO difference spectrum. The final traces are fit to asingle exponential using the software that is part of the instrument.Each experiment is done a minimum of six times, and the resulting ratesare averaged. The dissociation rates measured are independent ofdithionite concentration and independent of saturating CO as a trap forthe reduced species, both with and without 10 mM dithionite present.

Kinetic K_(D)

The kinetic K_(D) is determined by calculating the ratio of k_(off) tok_(on) using the measurements of k_(off) and k_(on) described above.

Calculated K_(D)

To measure the calculated K_(D), the values for the k_(off) and kineticK_(D) that are obtained as described above are graphed. A linearrelationship between k_(off) and kinetic K_(D) is defined by theequation (y=mx+b). k_(off) values were then interpolated along the lineto derive the calculated K_(D) using Excel: MAC 2004 (Microsoft,Redmond, Wash.). In the absence of a measured k_(on), this interpolationprovides a way to relate k_(off) to K_(D).

Rate of Autoxidation

To measure the rate of autoxidation, the protein samples areanaerobically reduced, then diluted to 5 μM heme in aerobic 50 mM TEA,50 mM NaCl, pH 7.5 buffer. These samples are then incubated in a Cary 3Espectrophotometer equipped with a Neslab RTE-100 constant-temperaturebath set to 37° C. and scanned periodically (Cary 3E, Varian, Inc., PaloAlto, Calif.). The rate of autoxidation is determined from thedifference between the maximum and minimum in the Fe^(III)-Fe^(II)difference spectrum plotted versus time and fit with a singleexponential using Excel: MAC 2004 (Microsoft, Redmond, Wash.).

Rate of Reaction with NO

NO reactivity is measured using purified proteins (H-NOX, polymericH-NOX, Homo sapiens hemoglobin (Hs Hb) etc.) prepared at 2 μM in bufferA and NO prepared at 200 μM in Buffer A (Buffer A: 50 mM Hepes, pH 7.5,50 mM NaCl). Data are acquired on a HI-TECH Scientific SF-61stopped-flow spectrophotometer equipped with a Neslab RTE-100constant-temperature bath set to 20° C. (TGK Scientific LTD., BradfordOn Avon, United Kingdom). The protein is rapidly mixed with NO in a 1:1ratio with an integration time of 0.00125 sec. The wavelengths ofmaximum change are fit to a single exponential using the software thatis part of the spectrometer, essentially measuring the rate-limitingstep of oxidation by NO. The end products of the reaction are ferric-NOfor the HNOX proteins and ferric-aquo for Hs Hb.

p50 Measurements

If desired, the p50 value for mutant or wild-type H-NOX proteins can bemeasured as described by Guarnone, R. et al. (September/October 1995).Haematologica 80(5):426-430, which is hereby incorporated by referencein its entirety, particularly with respect to the measurement of p50values. The p50 value is determined using a HemOx analyzer. Themeasurement chamber starts at 0% oxygen and slowly is raised,incrementally, towards 100% oxygen. An oxygen probe in the chambermeasures the oxygen saturation %. A second probe (UV-Vis light) measurestwo wavelengths of absorption, tuned to the alpha and beta peaks of thehemoprotein's (e.g., a protein such as H-NOX complexed with heme) UV-Visspectra. These absorption peaks increase linearly as hemoprotein bindsoxygen. The percent change from unbound to 100% bound is then plottedagainst the % oxygen values to generate a curve. The p50 is the point onthe curve where 50% of the hemoprotein is bound to oxygen.

Specifically, the Hemox-Analyzer (TCS Scientific Corporation, New Hope,Pa.) determines the oxyhemoprotein dissociation curve (ODC) by exposing50 μL of blood or hemoprotein to an increasing partial pressure ofoxygen and deoxygenating it with nitrogen gas. A Clark oxygen electrodedetects the change in oxygen tension, which is recorded on the x-axis ofan x-y recorder. The resulting increase in oxyhemoprotein fraction issimultaneously monitored by dual-wavelength spectrophotometry at 560 nmand 576 nm and displayed on the y-axis. Blood samples are taken from theantemedial vein, anticoagulated with heparin, and kept at 4° C. on wetice until the assay. Fifty μL of whole blood are diluted in 5 μL ofHemox-solution, a manufacturer-provided buffer that keeps the pH of thesolution at a value of 7.4-0.01. The sample-buffer is drawn into acuvette that is part of the Hemox-Analyzer and the temperature of themixture is equilibrated and brought to 37° C.; the sample is thenoxygenated to 100% with air. After adjustment of the pO₂ value thesample is deoxygenated with nitrogen; during the deoxygenation processthe curve is recorded on graph paper. The P50 value is extrapolated onthe x-axis as the point at which O₂ saturation is 50% using the softwarethat is part of the Hemox-Analyzer. The time required for a completerecording is approximately 30 minutes.

H-NOX Nucleic Acids

The invention also features nucleic acids encoding any of the mutantH-NOX proteins, polymeric H-NOX, or recombinant monomer H-NOX proteinsubunits as described herein.

In particular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any of nucleic acids encoding an H-NOXprotein or an H-NOX domain. In some embodiments, the nucleic acidincludes at least about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800,or more contiguous nucleotides from a H-NOX nucleic acid and containsone or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations)compared to the H-NOX nucleic acid from which it was derived. In variousembodiments, a mutant H-NOX nucleic acid contains less than about 20,15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the H-NOXnucleic acid from which it was derived. The invention also featuresdegenerate variants of any nucleic acid encoding a mutant H-NOX protein.

In some embodiments, the nucleic acid includes nucleic acids encodingtwo or more H-NOX domains. In some embodiments, the nucleic acidsincluding two or more H-NOX domains are linked such that a polymericH-NOX protein is expressed from the nucleic acid. In furtherembodiments, the nucleic acid includes nucleic acids encoding one ormore polymerization domains. In some embodiments, the nucleic acidsincluding the two or more H-NOX domains and the one or morepolymerization domains are linked such that a polymeric H-NOX protein isexpressed from the nucleic acid.

In some embodiments, the nucleic acid includes a segment or the entirenucleic acid sequence of any nucleic acid encoding a polymerizationdomain. In some embodiments the nucleic acid comprises a nucleic acidencoding an H-NOX domain and a polymerization domain. In someembodiments, the nucleic acid encoding an H-NOX domain and the nucleicacid encoding a polymerization domain a linked such that the producedpolypeptide is a fusion protein comprising an H-NOX domain and apolymerization domain.

In some embodiments, the nucleic acid comprises nucleic acid encodingone or more His₆ tags. In some embodiments the nucleic acid furthercomprised nucleic acids encoding linker sequences positioned betweennucleic acids encoding the H-NOX domain, the polymerization domainand/or a His₆ tag.

In some embodiments, the invention provides a nucleic acid encoding anH-NOX domain and a foldon domain. In some embodiments, the H-NOX domainis a T. thermoanaerobacter H-NOX domain. In some embodiments, the H-NOXdomain is a wild-type T. thermoanaerobacter H-NOX domain. In someembodiments, the H-NOX domain is a T. thermoanaerobacter L144F H-NOXdomain. In some embodiments, the H-NOX domain is a T. thermoanaerobacterW9F/L144F H-NOX domain.

In some embodiments, the invention provides nucleic acids encoding thefollowing 5′ to 3′: a L144F T. tengcongensis H-NOX domain, a Gly-Ser-Glyamino acid linker sequence, and a foldon domain. In some embodiments,the invention provides nucleic acids encoding the following 5′ to 3′: aW9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linkersequence, and a foldon domain. In some embodiments, the inventionprovides nucleic acids encoding the following 5′ to 3′: a wild-type T.tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence,and a foldon domain.

In some embodiments, the invention provides nucleic acids encoding thefollowing 5′ to 3′: a L144F T. tengcongensis H-NOX domain, a Gly-Ser-Glyamino acid linker sequence, a foldon domain, an Arg-Gly-Ser linkersequence, and a His₆ tag. In some embodiments, the invention providesnucleic acids encoding the following 5′ to 3′: a W9F/L144F T.tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, afoldon domain, an Arg-Gly-Ser linker sequence, and a His₆ tag. In someembodiments, the invention provides nucleic acids encoding the following5′ to 3′: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly aminoacid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence,and a His₆ tag.

In some embodiments, the nucleic acid comprises the nucleic acidsequence set forth in SEQ ID NO:5 or SEQ ID NO:7.

The invention also includes a cell or population of cells containing atleast one nucleic acid encoding a mutant H-NOX protein described herein.Exemplary cells include insect, plant, yeast, bacterial, and mammaliancells. These cells are useful for the production of mutant H-NOXproteins using standard methods, such as those described herein.

In some embodiments, the invention provides a cell comprising a nucleicacid encoding an H-NOX domain and a foldon domain. In some embodiments,the H-NOX domain is a T. thermoanaerobacter H-NOX domain. In someembodiments, the H-NOX domain is a wild-type T. thermoanaerobacter H-NOXdomain. In some embodiments, the H-NOX domain is a T. thermoanaerobacterL144F H-NOX domain. In some embodiments, the H-NOX domain is a T.thermoanaerobacter W9F/L144F H-NOX domain. In some embodiments, theinvention provides a cell comprising a nucleic acid comprising thenucleic acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.

Formulations of H-NOX Proteins

The present invention provides formulations of O₂ carrier polypeptidesfor use in enhancing tumor immunogenicity; for example, by inhibitingthe immune suppressive activities associated with tumor hypoxia. Anon-limiting exemplary family of O₂ carrier polypeptides is the H-NOXfamily of O₂ carrier polypeptides. Any wild-type or mutant H-NOXprotein, including polymeric H-NOX proteins, described herein may beused for the formulation of pharmaceutical or non-pharmaceuticalcompositions. In some embodiments, the formulations comprise a monomericH-NOX protein comprising an H-NOX domain and a polymerization domainsuch that the monomeric H-NOX proteins associate in vitro or in vivo toproduce a polymeric H-NOX protein. As discussed further below, theseformulations are useful in a variety of therapeutic and industrialapplications.

In some embodiments, the pharmaceutical composition includes one or morewild-type or mutant H-NOX proteins described herein including polymericH-NOX proteins and a pharmaceutically acceptable carrier or excipient.Examples of pharmaceutically acceptable carriers or excipients include,but are not limited to, any of the standard pharmaceutical carriers orexcipients such as phosphate buffered saline solutions, water, emulsionssuch as oil/water emulsion, and various types of wetting agents.Exemplary diluents for aerosol or parenteral administration arephosphate buffered saline or normal (0.9%) saline. Compositionscomprising such carriers are formulated by well-known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; andRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000, which are each hereby incorporated by reference intheir entireties, particularly with respect to formulations). In someembodiments, the formulations are sterile. In some embodiments, theformulations are essentially free of endotoxin.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions can be formulated for any appropriate manner ofadministration, including, for example, intravenous, intra-arterial,intravesicular, intratumoral, inhalation, intraperitoneal,intrapulmonary, intramuscular, subcutaneous, intra-tracheal,transmucosal, intraocular, intrathecal, or transdermal administration.For parenteral administration, such as subcutaneous injection, thecarrier may include, e.g., water, saline, alcohol, a fat, a wax, or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, or magnesium carbonate,may be employed. Biodegradable microspheres (e.g., polylactatepolyglycolate) may also be used as carriers.

In some embodiments, the pharmaceutical or non-pharmaceuticalcompositions include a buffer (e.g., neutral buffered saline, phosphatebuffered saline, etc), a carbohydrate (e.g., glucose, mannose, sucrose,dextran, etc.), an antioxidant, a chelating agent (e.g., EDTA,glutathione, etc.), a preservative, another compound useful for bindingand/or transporting oxygen, an inactive ingredient (e.g., a stabilizer,filler, etc.), or combinations of two or more of the foregoing. In someembodiments, the composition is formulated as a lyophilizate. H-NOXproteins may also be encapsulated within liposomes or nanoparticlesusing well known technology. Other exemplary formulations that can beused for H-NOX proteins are described by, e.g., U.S. Pat. Nos.6,974,795, and 6,432,918, which are each hereby incorporated byreference in their entireties, particularly with respect to formulationsof proteins.

The compositions described herein may be administered as part of asustained release formulation (e.g., a formulation such as a capsule orsponge that produces a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain an H-NOX proteindispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane. Carriers for use within suchformulations are biocompatible, and may also be biodegradable. In someembodiments, the formulation provides a relatively constant level ofH-NOX protein release. The amount of H-NOX protein contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release, and the nature of the conditionto be treated or prevented.

In some embodiments, the pharmaceutical composition contains aneffective amount of a wild-type or mutant H-NOX protein. In someembodiments, the pharmaceutical composition contains an effective amountof a polymeric H-NOX protein comprising two or more wild-type or mutantH-NOX domains. In some embodiments, the pharmaceutical compositioncontains an effective amount of a recombinant monomeric H-NOX proteincomprising a wild-type or mutant H-NOX domain and a polymerizationdomain as described herein. In some embodiments, the formulationcomprises a trimeric H-NOX protein comprising three monomers, eachmonomer comprising a T. tengcongensis L144F H-NOX domain and a foldondomain. In some embodiments, the formulation comprises a trimeric H-NOXprotein comprising three monomers, each monomer comprising a T.tengcongensis W9F/L144F H-NOX domain and a foldon domain. In someembodiments, the formulation comprises a trimeric H-NOX proteincomprising three monomers, each monomer comprising a T. tengcongensisL144F H-NOX domain and a foldon domain. In some embodiments, theformulation comprises a PEGylated trimeric H-NOX protein comprisingthree monomers, each monomer comprising a T. tengcongensis L144F H-NOXdomain and a foldon domain. In some embodiments, the pharmaceuticalcomposition comprises an O₂ carrier polypeptide (e.g., an H-NOX protein)in an amount effective to modulate tumor immunity (e.g., enhance animmune response to the tumor).

In some embodiments, an effective amount of an H-NOX protein foradministration to a human is between a few grams to over about 350grams. Other exemplary doses of an H-NOX protein include about any of4.4., 5, 10, or 13 G/DL (where G/DL is the concentration of the H-NOXprotein solution prior to infusion into the circulation) at anappropriate infusion rate, such as about 0.5 ml/min (see, for example,Winslow, R. Chapter 12 In Blood Substitutes). It will be appreciatedthat the unit content of active ingredients contained in an individualdose of each dosage form need not in itself constitute an effectiveamount since the necessary effective amount could be reached by thecombined effect of a plurality of administrations. The selection of theamount of an H-NOX protein to include in a pharmaceutical compositiondepends upon the dosage form utilized, the condition being treated, andthe particular purpose to be achieved according to the determination ofthe ordinarily skilled artisan in the field.

Exemplary compositions include genetically engineered, recombinant H-NOXproteins, which may be isolated or purified, comprising one or moremutations that collectively impart altered O₂ or NO ligand-bindingrelative to the corresponding wild-type H-NOX protein, and operative asa physiologically compatible mammalian blood gas carrier. For example,mutant H-NOX proteins as described herein. In some embodiments, theH-NOX protein is a polymeric H-NOX protein. In some embodiments, theH-NOX protein is a recombinant monomeric H-NOX protein comprising awild-type or mutant H-NOX domain and a polymerization domain asdescribed herein. In some embodiments, the composition comprises atrimeric H-NOX protein comprising three monomers, each monomercomprising a T. tengcongensis L144F H-NOX domain and a foldon domain. Insome embodiments, the composition comprises a trimeric H-NOX proteincomprising three monomers, each monomer comprising a T. tengcongensisW9F/L144F H-NOX domain and a foldon domain. In some embodiments, thecomposition comprises a trimeric H-NOX protein comprising threemonomers, each monomer comprising a T. tengcongensis L144F H-NOX domainand a foldon domain. In some embodiments, the composition comprises aPEGylated trimeric H-NOX protein comprising three monomers, each monomercomprising a T. tengcongensis L144F H-NOX domain and a foldon domain.

To reduce or prevent an immune response in human subjects who areadministered a pharmaceutical composition, human H-NOX proteins ordomains (either wild-type human proteins or human proteins into whichone or more mutations have been introduced) or other non-antigenic H-NOXproteins or domains (e.g., mammalian H-NOX proteins) can be used. Toreduce or eliminate the immunogenicity of H-NOX proteins derived fromsources other than humans, amino acids in an H-NOX protein or H-NOXdomain can be mutated to the corresponding amino acids in a human H-NOX.For example, one or more amino acids on the surface of the tertiarystructure of a non-human H-NOX protein can be mutated to thecorresponding amino acid in a human H-NOX protein.

Methods to Modulate Tumor Immunity

In some aspects, the invention provides methods modulate to tumorimmunity and thus can be used in anticancer treatments. Hypoxic tumormicroenvironments suppress the host's immune anti-tumor defenses bymodulating multiple signaling pathways (FIG. 1) including, but notlimited to, hypoxia inducible factor (HIF-1) signaling (Codo et al.,2014 Oncotarget, 5(17), 7651-7662; Lee, Mace, & Repasky, 2010 Int JHyperthermia, 26(3), 232-246; Wei et al., 2011 PLoS One, 6(1), e16195),miRNA epigenetic regulation of antitumor T cells, MHC1 expression ontumor cells, and recruitment of tumor associated macrophages andmyeloid-derived suppressor cells (MDSC). Hypoxic activation of the HIF-1pathway has been shown to activate adenosinergic A2 and PD-L1 pathwayswhich in turn inhibit recruitment and activation of helper and killerT-cells and NK cells (Noman et al., 2014 J Exp Med, 211(5), 781-790;Ohta et al., 2006 Proc Natl Acad Sci USA, 103(35), 13132-13137). Hypoxicactivation of the HIF-1 pathway may also lead to the recruitment andactivation of inhibitory regulatory T cells (Treg), tumor associatedmacrophages (TAM) and other myeloid-derived suppressor cells (MDSC)(Chaturvedi et al., 2014 Proc Natl Acad Sci USA, 111(20), E2120-2129;Corzo et al., 2010 J Exp Med, 207(11), 2439-2453; Wei et al., 2011).HIF-1 pathway activation may also directly inhibit the ability of tumorcells to be recognized by immune system by increasing tumor shedding ofMHC1 receptors (Siemens et al., 2008 Cancer Res, 68(12), 4746-4753).

In some aspects, the invention provides methods for modulating tumorimmunity in an individual with a tumor comprising administering to theindividual an effective amount of an O₂ carrier polypeptide (e.g., anH-NOX protein). In some embodiments, the modulating of tumor immunitycomprises enhancing an immune response to the tumor. In someembodiments, the invention provides methods for increasing leucocyteinfiltration to a tumor in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide. In someembodiments, the invention provides methods for increasing lymphocyteinfiltration to a tumor in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide. In someembodiments, the increase in lymphocyte infiltration to the tumorcomprises an increase in infiltration of one or more of CD4 cells, CD8cells, or NK cells. In some embodiments, the modulating of tumorimmunity comprises increasing antigen processing. In some embodiments,the modulating of tumor immunity comprises increasing the presentationcapabilities of dendritic cells (DC). In some embodiments, themodulating of tumor immunity comprises one or more of increasinglymphocyte infiltration to the tumor, increasing antigen processing, orincreasing DC presentation capability. In some embodiments, themodulating of tumor immunity comprises lymphocyte activation. In someembodiments, the modulating of tumor immunity comprises cytokinesecretion. In some embodiments, the O₂ carrier polypeptide is a trimericTt H-NOX L144F polypeptide. In some embodiments, the O₂ carrierpolypeptide is a PEGylated trimeric Tt H-NOX L144F polypeptide.

In some embodiments of the invention, the increase in lymphocyteinfiltration to the tumor is accompanied by inhibition of one or more ofTreg cells, tumor associated macrophages or myeloid derived suppressorcells in the tumor. In some embodiments, the increase in lymphocyteinfiltration to the tumor is accompanied by an increase in MHC1expression on the tumor cells.

In some embodiments, the invention provides methods for decreasingexpression of HIF-1α in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide (e.g. an H-NOX protein). In some embodiments, administrationof an effective amount of an O₂ carrier polypeptide (e.g., an H-NOXprotein) to an individual results in a decrease in expression of HIF-1α.In some embodiments, the expression of HIF-1α is decreased by more thanabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to expression of HIF-1α in the absence of treatment with an O₂carrier polypeptide. In some embodiments, the expression of HIF-1α isreduced compared to expression of HIF-1α in the absence of treatmentwith an O₂ carrier protein for more than about any of 1 hr, 2 hr, 3 hr,4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr42 hr or 48 hr. In some embodiments, the O₂ carrier polypeptide is atrimeric Tt H-NOX L144F polypeptide. In some embodiments, the O₂ carrierpolypeptide is a PEGylated trimeric Tt H-NOX L144F polypeptide.

In some embodiments, the invention provides methods for decreasingexpression of HIF-1α in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide (e.g. an H-NOX protein) wherein the decrease in expressionof HIF-1α is measured as a decrease in expression of vascular epithelialcell growth factor (VEGF). In some embodiments, administration of aneffective amount of an O₂ carrier polypeptide (e.g., an H-NOX protein)to an individual results in a decrease in expression of VEGF. In someembodiments, the expression of VEGF is decreased by more than about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared toexpression of VEGF in the absence of treatment with an O₂ carrierpolypeptide. In some embodiments, the expression of VEGF is reducedcompared to expression of VEGF in the absence of treatment with an O₂carrier protein for more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr,6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48hr. In some embodiments, the O₂ carrier polypeptide is a trimeric TtH-NOX L144F polypeptide. In some embodiments, the O₂ carrier polypeptideis a PEGylated trimeric Tt H-NOX L144F polypeptide.

In some embodiments, the invention provides methods for decreasingexpression of HIF-1α in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide (e.g. an H-NOX protein) herein the decrease in expression ofHIF-1α is measured as a decrease in expression of glucose transportertype 1 (Glut1). In some embodiments, administration of an effectiveamount of an O₂ carrier polypeptide (e.g., an H-NOX protein) to anindividual results in a decrease in expression of Glut1. In someembodiments, the expression of Glut1 is decreased by more than about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared toexpression of Glut1 in the absence of treatment with an O₂ carrierpolypeptide. In some embodiments, the expression of Glut1 is reducedcompared to expression of Glut1 in the absence of treatment with an O₂carrier protein for more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr,6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48hr. In some embodiments, the O₂ carrier polypeptide is a trimeric TtH-NOX L144F polypeptide. In some embodiments, the O₂ carrier polypeptideis a PEGylated trimeric Tt H-NOX L144F polypeptide.

In some embodiments, the invention provides methods for decreasingexpression of PD-L1 in a tumor in an individual comprising administeringto the individual an effective amount of an O₂ carrier polypeptide (e.g.an H-NOX protein). In some embodiments, administration of an effectiveamount of an O₂ carrier polypeptide (e.g., an H-NOX protein) to anindividual results in a decrease in expression of PD-L1. In someembodiments, the expression of PD-L1 is decreased by more than about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared toexpression of PD-L1 in the absence of treatment with an O₂ carrierpolypeptide. In some embodiments, administration of an effective amountof an O₂ carrier polypeptide (e.g., an H-NOX protein) to an individualresults in a decrease in the interaction of PD-L1 with PD-1. In someembodiments, the interaction of PD-L1 with PD1 is decreased by more thanabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to interaction of PD-L1 with PD1 in the absence of treatmentwith an O₂ carrier polypeptide. In some embodiments, the expression ofPD-L1 is reduced compared to expression of PD-L1 in the absence oftreatment with an O₂ carrier protein for more than about any of 1 hr, 2hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30hr, 36, hr 42 hr or 48 hr. In some embodiments, the O₂ carrierpolypeptide is a trimeric Tt H-NOX L144F polypeptide. In someembodiments, the O₂ carrier polypeptide is a PEGylated trimeric Tt H-NOXL144F polypeptide.

In some embodiments, the invention provides methods for decreasingexpression of A2AR in a tumor in an individual comprising administeringto the individual an effective amount of an O₂ carrier polypeptide (e.g.an H-NOX protein). In some embodiments, administration of an effectiveamount of an O₂ carrier polypeptide (e.g., an H-NOX protein) to anindividual results in a decrease in expression of A2AR. In someembodiments, the expression of A2AR is decreased by more than about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared toexpression of A2AR in the absence of treatment with an O₂ carrierpolypeptide. In some embodiments, administration of an effective amountof an O₂ carrier polypeptide (e.g., an H-NOX protein) to an individualresults in a decrease in the interaction of A2AR with adenosine. In someembodiments, the interaction of A2AR with adenosine is decreased by morethan about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to interaction of A2AR with adenosine in the absence oftreatment with an O₂ carrier polypeptide. In some embodiments, theexpression of A2AR is reduced compared to expression of A2AR in theabsence of treatment with an O₂ carrier protein for more than about anyof 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr,24 hr, 30 hr, 36, hr 42 hr or 48 hr. In some embodiments, the O₂ carrierpolypeptide is a trimeric Tt H-NOX L144F polypeptide. In someembodiments, the O₂ carrier polypeptide is a PEGylated trimeric Tt H-NOXL144F polypeptide.

In some embodiments, the invention provides methods for decreasingexpression of HIF-2α in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide (e.g. an H-NOX protein). In some embodiments, administrationof an effective amount of an O₂ carrier polypeptide (e.g., an H-NOXprotein) to an individual results in a decrease in expression of HIF-2α.In some embodiments, the expression of HIF-2α is decreased by more thanabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%compared to expression of HIF-2α in the absence of treatment with an O₂carrier polypeptide. In some embodiments, administration of an effectiveamount of an O₂ carrier polypeptide (e.g., an H-NOX protein) to anindividual results in a decrease in the expression of HIF-2u. In someembodiments, the expression of HIF-2u is decreased by more than aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared toexpression of HIF-2α in the absence of treatment with an O₂ carrierpolypeptide. In some embodiments, the expression of HIF-2α is reducedcompared to expression of HIF-2α in the absence of treatment with an O₂carrier protein for more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr,6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48hr. In some embodiments, the O₂ carrier polypeptide is a trimeric TtH-NOX L144F polypeptide. In some embodiments, the O₂ carrier polypeptideis a PEGylated trimeric Tt H-NOX L144F polypeptide.

In some embodiments, the invention provides methods for modulating tumorimmunity (e.g., enhancing an immune response to a tumor) in anindividual by any of the methods described herein. Examples of tumorsinclude but are not limited to a brain tumor, a glioblastoma, a bonetumor, a pancreatic tumor, a skin tumor, a tumor of the head or neck, amelanoma, a lung tumor, a uterine tumor, an ovarian tumor, a colorectaltumor, an anal tumor, a liver tumor, a hepatocellular carcinoma, astomach tumor, a testicular tumor, an endometrial tumor, a cervicaltumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma,an esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenaltumor, a bladder tumor, a kidney tumor, a breast tumor, a multiplemyeloma tumor, a sarcoma, or a squamous cell tumor.

In some embodiments, the invention provides methods for modulating tumorimmunity (e.g., enhancing an immune response to a tumor) in anindividual by any of the methods described herein thereby providingmethods for treating cancer in an individual. Examples of cancers thatmay be treated by the methods of the invention include but are notlimited to brain cancer, glioblastoma, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, melanoma, lung cancer, uterinecancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer,hepatocellular carcinoma, stomach cancer, testicular cancer, endometrialcancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma,esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer,bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma,or squamous cell cancer.

In some embodiments, the invention provides methods for modulating tumorimmunity in an individual by any of the methods described herein. Insome embodiments, the individual is a mammal; for example a human. Insome embodiments, the mammal is a pet, a laboratory research animal, ora farm animal. Non-limiting examples of pets, research animals or farmanimals include dogs, cats, horses, monkeys, rabbits, rats, mice, guineapigs, hamsters, pigs and cows.

O₂ carrier polypeptides may be administered by any route including butnot limited to intravenous, intra-arterial, intratumoral,intravesicular, inhalation, intraperitoneal, intrapulmonary,intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular,intrathecal, or transdermal administration.

In some aspects, sustained delivery of oxygen to a tumor is desired toinhibit hypoxia-mediated tumor immunity and to enhance an immuneresponse to the tumor. In some embodiments of the invention,administration of the O₂ carrier polypeptide (e.g., H-NOX protein) isrepeated. Administration of the O₂ carrier polypeptide may be repeateduntil a robust immune response to the tumor is established. In someembodiments, administration of the O₂ carrier polypeptide is repeated atleast about any one of two times, three times, four times, five times,six times, seven times, eight times, nine times, ten times, twelvetimes, fourteen times, twenty times, thirty times, forty times, fiftytimes or one hundred times. In some embodiments, administration of theO₂ carrier polypeptide is repeated between about two times and abouttwenty times. In some embodiments, administration of the O₂ carrierpolypeptide is repeated between any one of about twenty times and aboutforty times, any one of about forty times and about sixty times, any oneof about sixty times and about eighty times, any one of about eightytimes and about one hundred times, or any number of times therebetween.In some embodiments, administration of the O₂ carrier polypeptide isrepeated daily or twice a day for about 42 to about 84 administrations.

Exemplary dosing frequencies include, but are not limited to, at least1, 2, 3, 4, 5, 6, or 7 times (i.e., daily) a week. In some embodiments,the O₂ carrier polypeptide (e.g., H-NOX protein) is administered atleast 2, 3, 4, or 6 times a day. In some embodiments, the O₂ carrierpolypeptide is administered every four, every 8, every 12, every 24hours, every 48 hours or two times a week or three times a week. In someembodiments, the O₂ carrier polypeptide is administered any one ofbetween one hour and two hours, between two hours and four hours,between four hours and eight hours, between eight hours and twelvehours, or between twelve hours and 24 hours. In some embodiments, the O₂carrier polypeptide is administered every four, every 8, every 12 orevery 24 hours for a period of about one to about 10 days. In someembodiments, the O₂ carrier polypeptide can be administered, e.g., overa period of a few days or weeks. In some embodiments, the O₂ carrierpolypeptide is administrated for a longer period, such as a few monthsor years. The dosing frequency of the composition may be adjusted overthe course of the treatment based on the judgment of the administeringphysician.

In some embodiments, the O₂ carrier polypeptide (e.g., H-NOX protein) isadministered as a bolus. In some embodiments, the volume of the bolus isgreater than about any of 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 50 mL, 75 mL, or 100 mL. In someembodiments, administration of the bolus dose is repeated as above.

In some embodiments, the O₂ carrier polypeptide (e.g., H-NOX protein) isadministered by infusion. In some embodiments, the infusions is forgreater than about any of 15 minutes, 30 minutes, 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 16 hours, 20 hours or 24 hours. In some embodiments, theinfusions is for between about any of 15 minutes and 30 minutes, 30minutes and 1 hour, 1 hour and 2 hours, 2 hours and 3 hours, 3 hours and4 hours, 4 hours and 5 hours, 5 hours and 6 hours, 6 hours and 7 hours,7 hours and 8 hours, 8 hours and 9 hours, 9 hours and 10 hours, 10 hoursand 12 hours, 12 hours and 16 hours, 16 hours and 20 hours or 20 hoursand 24 hours. In some embodiments, the infusion rate is greater any ofabout 1 mL/hr, 2 mL/hr, 3 mL/hr, 4 mL/hr, 5 mL/hr, 6 mL/hr, 7 mL/hr, 8mL/hr, 9 mL/hr, 10 mL/hr, 20 mL/hr, 30 mL/hr, 40 mL/hr, 50 mL/hr, 60mL/hr, 70 mL/hr, 80 mL/hr, 90 mL/hr, 100 mL/hr, 200 mL/hr, 300 mL/hr,400 mL/hr, 500 mL/hr, 600 mL/hr, 700 mL/hr, 800 mL/hr, 900 mL/hr, 1000mL/hr, 2000 mL/hr, 3000 mL/hr, 4000 mL/hr, 5000 mL/hr, 6000 mL/hr, 7000mL/hr, 8000 mL/hr, 9000 mL/hr, 10,000 mL/hr or any rate therebetween. Insome embodiments, the infusion is repeated as above.

In some embodiments, the O₂ carrier polypeptide (e.g., H-NOX protein) isadministered at a dose of greater than about any of 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg,15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg,500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, orany dose therebetween. In some embodiments, the dose is provided as oneor more bolus administrations.

In some embodiments, the dose is provided as one or more infusions. Insome embodiments the dose is provided in more than one administration(e.g., a dose of 100 mg/kg may be provided by two doses of 50 mg/kg).

In some embodiments of the invention, the O₂ carrier polypeptide (e.g.an H-NOX protein) is used in combination with radiation therapy. In someembodiments, the O₂ carrier polypeptide is administered to theindividual any of at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20,22, or 24 hours before administration of the radiation. In someembodiments, the radiation is X irradiation. In some embodiments, thedose of X irradiation is any of about 0.5 Gy to about 75 Gy. In someembodiments, the cycle of O₂ carrier polypeptide administration andradiation administration is repeated any one of one, two, three, four,five or six times. In some embodiments, the cycle of O₂ carrierpolypeptide administration and radiation administration is repeatedafter any one of about one week, two weeks, three weeks, four weeks,five weeks or six weeks. In some embodiments, the administration of theO₂ carrier polypeptide and radiation therapy is used in conjunction withanother therapy; for example, a chemotherapy and/or immunotherapy.

In some embodiments of the invention, the O₂ carrier polypeptide (e.g.an H-NOX protein) is used in combination with chemotherapy. In someembodiments, the chemotherapy is a cytotoxin. Chemotherapeutic agentsincluding cytotoxins are known in the art. In some embodiments, thecytotoxin is an alkylating agent. In some embodiments, the cytotoxin iscyclophosphamide or temozolomide. In some embodiments, the O₂ carrierpolypeptide is administered before administration of the chemotherapy.In some embodiments, the O₂ carrier polypeptide is administered withadministration of the chemotherapy. In some embodiments, the O₂ carrierpolypeptide is administered after administration of the chemotherapy. Insome embodiments, the O₂ carrier polypeptide is administered to theindividual any of at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18,20, 22, or 24 hours or is administered daily or twice a day for 1, 2, 3,4, 5, 6, or 7 days before administration of the chemotherapy. In someembodiments, the O₂ carrier polypeptide is administered to theindividual any of at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, or 24 hoursafter administration of the chemotherapy. In some embodiments,administration of the O₂ carrier polypeptide and/or administration ofthe chemotherapy is repeated any one of one, two, three, four, five,six, seven, eight, nine, ten times or more than ten times. In someembodiments, administration of the O₂ carrier polypeptide and/oradministration of the chemotherapy is repeated after any one of aboutone week, two weeks, three weeks, four weeks, five weeks or six weeks.In some embodiments, administration of the O₂ carrier polypeptide andthe chemotherapy are on the same dosing cycle. In some embodiments,administration of the O₂ carrier polypeptide and the chemotherapy are ondifferent dosing cycles. In some embodiments, the admiration of H-NOXand radiation therapy is used in conjunction with another therapy; forexample, radiation therapy and/or immunotherapy.

In some embodiments, the O₂ carrier polypeptide (e.g., an H-NOX protein)is administered to cancer patients prior to and/or in conjunction withan immunotherapy. In some embodiments, the immunotherapy is one or moreof an anticancer vaccine, an adoptive immune cell therapy, an agent thattargets an immune checkpoint regulator, an oncolytic virus or a BiTE. Insome embodiments, the immunotherapy targets are one or more of CTLA-4,PD1, PD-L1, or an immune checkpoint regulator. In some embodiments, theimmunotherapy is a dual PD1/CTLA-4 blockade therapy. In someembodiments, the immunotherapy is a PDL-1 treatment for patients withPDL1+ tumors or dual PD1/PD-L1 blockade. Nonlimiting examples includebut are not limited to PD-1 and PDL-1 antagonists such as antibodies(e.g., Nivolumab). In some embodiments the checkpoint inhibitor is aCTLA4 antagonist such as an antibody (e.g., ipilumumab). In someembodiments, the immunotherapy is an adoptive T cell therapy includingbut not limited to chimeric antigen receptor T cells (e.g., CAR-T cells)or engineered TCR-T cells. In some embodiments, the immunotherapy is aBispecific T cell Engagers (BiTE). In some embodiments, theimmunotherapy includes one or more of anti-lymphocyte activation gene3(LAG-3) therapy, anti-T cell immunoglobin mucin-3 (TIM-3) therapy,anti-killer immunoglobin-like receptors (KIR) therapy, anti-4-1BB(CD137) agonizing/stimulatory therapy, or glucocorticoid-induced TNFRfamily related gene (GITR) agonizing/stimulatory therapy—each alone orin combinations with each other, and/or in combination with one or moreof PD1, PDL-1, CTLA-4 or other therapies.

Nonlimiting examples of therapies that target checkpoint proteins otherthan PD-1/PDL-1 and CTLA4 negative regulators include both positive andnegative (checkpoint inhibitors) regulators of immune response and canbe antibodies or small molecules such as IDO (indoleamine-2.3dioxygenase) pathway inhibitors such as direct IDO enzymatic activityinhibitors (e.g. NLG919), IDO effector pathway inhibitors (e.g.D-1-methyl-tryptophan, Indoximod, NLG8189), TDO (tryptophan2,3-dioxygenase) inhibitors, or IDO-TDO dual inhibitors;Lymphocyte-activation gene 3 (LAG-3, CD223) antibody antagonists (e.g.IMP321, BMS-986016); Killer immunoglobulin-like receptors (KIRs)antagonists such as antibodies (e.g. lirilumab, IPH2101); T cellimmunoglobulin mucin-3 (TIM-3) antagonists such as antibodies; B- and Tcell attenuator (BTLA, CD272) antagonists such as antibodies; OX40(CD134) agonists such as activating/stimulating antibodies; 4-1BB(CD137) agonists such as stimulatory antibodies (e.g. BMS-663513);Glucocorticoid-induced TNFR family related gene (GITR) agonists such asstimulatory antibodies (e.g. TRX518); and oncolytic viruses.

In some embodiments, the O₂ carrier polypeptide is administered beforeadministration of the immunotherapy. In some embodiments, the O₂ carrierpolypeptide is administered with administration of the immunotherapy. Insome embodiments, the O₂ carrier polypeptide is administered afteradministration of the immunotherapy. In some embodiments, the O₂ carrierpolypeptide is administered to the individual any of at least 1, 2, 3,4, 5, 6, 7, 8, 10, 12, 24, or 48 hours before administration of theimmunotherapy. In some embodiments, the O₂ carrier polypeptide isadministered to the individual any of at least 3, 4, 5, 6, 7 or moredays before administration of the immunotherapy. In some embodiments,the O₂ carrier polypeptide is administered to the individual any of atleast 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24 or 48 hours afteradministration of the immunotherapy. In some embodiments, the O₂ carrierpolypeptide is administered to the individual any of at least 3, 4, 5,6, 7 or more days after administration of the immunotherapy. In someembodiments, administration of the O₂ carrier polypeptide and/oradministration of the immunotherapy is repeated any one of one, two,three, four, five, six, seven, eight, nine, ten times or more than tentimes. In some embodiments, administration of the O₂ carrier polypeptideand/or administration of the immunotherapy is repeated after any one ofabout one week, two weeks, three weeks, four weeks, five weeks or sixweeks. In some embodiments, administration of the O₂ carrier polypeptideand the immunotherapy are on the same dosing cycle. In some embodiments,administration of the O₂ carrier polypeptide and the immunotherapy areon different dosing cycles. In some embodiments, the admiration of H-NOXand radiation therapy is used in conjunction with another therapy; forexample, radiation therapy and/or chemotherapy.

In some embodiments, the effectiveness of administration of the O₂carrier polypeptide (e.g., H-NOX protein) is monitored; for example butnot limited to tumor hypoxia, expression hypoxia-associated tumorsuppressors and/or activators, presence of tumor-associated immune cellsand/or immune cells directed against tumor cells and/or local (tumorbiopsy, lymph node biopsy) or systemic (e.g. peripheral blood) cytokineand immune cell profiles. Methods to determine the level of tumorhypoxia are known in the art. Examples include but are not limited tomeasurement of any one of ¹⁸F-fluoromisonidazole (FMISO) tumor uptake,pimidazole uptake, ¹⁸F-fluoroazomycin arabinoside (FAZA) uptake, anitroimidazole uptake,Copper(II)-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM) uptake,hexafluorobenzene (C6F6) uptake by ¹⁹F magnetic resonance imaging,hexamethyldisiloxane uptake by ¹H MRI, tumor HIF-1α expression, tumorHIF-2α expression, tumor HIF-3α expression, tumor Glut-1 expression,tumor pH (pH-weighted MRI) qBOLD, OE-MRI, MOBILE MRI tumor LDHAexpression, tumor carbonic anhydrase IX (CA-9) expression, VEGFexpression, or lactate and/or pyruvate levels. In some embodiments ofthe methods of monitoring, treating, and optimization of therapydescribed above, tumor hypoxia is measured by ¹⁸F-FMISO uptake. In someembodiments, ¹⁸F-FMISO uptake is measured by Positron emissiontomography (PET) scan, computed tomography (CT) scan or computed axialtomography (CAT) scan. Methods to detect expression of genes such asHIF-1α, PD-L1 and A2AR are known in the art; for example, byimmunoassay, by immunohistochemistry, by quantitative PCR, byhybridization (for example, on a gene chip), and the like.

Kits with H-NOX Proteins

Also provided are articles of manufacture and kits for the modulation oftumor immunity in an individual. In some embodiments, the article ofmanufacture or kit comprises any of the O₂ carrier polypeptidesincluding any of the H-NOX proteins described herein including polymericH-NOX proteins and PEGylated polymeric H-NOX proteins, and suitablepackaging. In some embodiments, the invention includes a kit with (i) aH-NOX protein (such as a wild-type or mutant H-NOX protein describedherein or formulations thereof as described herein) and (ii)instructions for using the kit to deliver O₂ to an individual.

Suitable packaging for compositions described herein are known in theart, and include, for example, vials (e.g., sealed vials), vessels,ampules, bottles, jars, flexible packaging (e.g., sealed Mylar orplastic bags), and the like. These articles of manufacture may furtherbe sterilized and/or sealed. Also provided are unit dosage formscomprising the compositions described herein. These unit dosage formscan be stored in a suitable packaging in single or multiple unit dosagesand may also be further sterilized and sealed. Instructions supplied inthe kits of the invention are typically written instructions on a labelor package insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable. The instructions relatingto the use of H-NOX proteins generally include information regardingdosage, dosing schedule, and route of administration for the intendedtreatment or industrial use. The kit may further comprise a descriptionof selecting an individual suitable for treatment.

The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or sub-unit doses. For example, kits may also be provided thatcontain sufficient dosages of H-NOX proteins disclosed herein to provideeffective treatment for an individual for an extended period, such asabout any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, ormore. Kits may also include multiple unit doses of H-NOX proteins andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies. In some embodiments, the kit includes a dry (e.g.,lyophilized) composition that can be reconstituted, resuspended, orrehydrated to form generally a stable aqueous suspension of H-NOXprotein.

Exemplary Methods for Production of H-NOX Proteins

As noted above, the sequences of several wild-type H-NOX proteins andnucleic acids are known and can be used to generate mutant H-NOX domainsand nucleic acids of the present invention. Techniques for the mutation,expression, and purification of recombinant H-NOX proteins have beendescribed by, e.g., Boon, E. M. et al. (2005). Nature Chemical Biology1:53-59 and Karow, D. S. et al. (Aug. 10, 2004). Biochemistry43(31):10203-10211, U.S. Pat. Nos. 8,404,631 and 8,404,632, WO2007/139791, and WO 2007/139767 which are hereby incorporated byreference in their entireties, particularly with respect to themutation, expression, and purification of recombinant H-NOX proteins.These techniques or other standard techniques can be used to generateany mutant H-NOX protein.

A mutant H-NOX nucleic acid can be incorporated into a vector, such asan expression vector, using standard techniques. For example,restriction enzymes can be used to cleave the mutant H-NOX nucleic acidand the vector. Then, the compatible ends of the cleaved mutant H-NOXnucleic acid and the cleaved vector can be ligated. The resulting vectorcan be inserted into a cell (e.g., an insect cell, a plant cell, a yeastcell, or a bacterial cell) using standard techniques (e.g.,electroporation) for expression of the encoded H-NOX protein.

In particular, heterologous proteins have been expressed in a number ofbiological expression systems, such as insect cells, plant cells, yeastcells, and bacterial cells. Thus, any suitable biological proteinexpression system can be utilized to produce large quantities ofrecombinant H-NOX protein. In some embodiments, the H-NOX protein (e.g.,a mutant or wild-type H-NOX protein) is an isolated protein.

If desired, H-NOX proteins can be purified using standard techniques. Insome embodiments, the protein is at least about 60%, by weight, freefrom other components that are present when the protein is produced. Invarious embodiments, the protein is at least about 75%, 90%, or 99%, byweight, pure. A purified protein can be obtained, for example, bypurification (e.g., extraction) from a natural source, a recombinantexpression system, or a reaction mixture for chemical synthesis.Exemplary methods of purification include immunoprecipitation, columnchromatography such as immunoaffinity chromatography, magnetic beadimmunoaffinity purification, and panning with a plate-bound antibody, aswell as other techniques known to the skilled artisan. Purity can beassayed by any appropriate method, e.g., by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. In someembodiments, the purified protein is incorporated into a pharmaceuticalcomposition of the invention or used in a method of the invention. Thepharmaceutical composition of the invention may have additives,carriers, or other components in addition to the purified protein.

In some embodiments, the polymeric H-NOX protein comprises one or moreHis₆ tags. An H-NOX protein comprising at least one His₆ tag may bepurified using chromatography; for example, using Ni²⁺-affinitychromatography. Following purification, the His₆ tag may be removed; forexample, by using an exopeptidase. In some embodiments, the inventionprovides a purified polymeric H-NOX protein, wherein the polymeric H-NOXprotein was purified through the use of a His₆ tag. In some embodiments,the purified H-NOX protein is treated with an exopeptidase to remove theHis₆ tags.

In some embodiments, H-NOX protein comprises one or more molecules ofpolyethylene glycol (i.e., PEGylated). Methods to produce PEGylatedproteins are known in the art.

Examples

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The examples are not intended to represent that theexperiments below are all or the only experiments performed. Unlessindicated otherwise, temperature is in degrees Centigrade and pressureis at or near atmospheric.

Example 1. H-NOX Enables Efficient Oxygenation of Hypoxic TumorMicroenvironments

A PEGylated trimeric Thermoanaerobacter tengcongensis (Tt.) H-NOXbearing a L144F substitution in the distal pocket (FIG. 6B) wasevaluated for the ability to oxygenate tumor microenvironments andincrease radiation sensitity. Administration of the PEGylated trimericTt H-NOX L144F to mice bearing hypoxic tumors induces rapid andsustained oxygenation of the tumors as directly measured by the externalhypoxia marker, pimonidazole, hypoxia inducible transcription factor 1alpha, HIF-1-α, and OxyLite oxygen-sensing nanofiber (FIGS. 2 and 3,respectively).

Mice bearing H460 subcutaneous xenograft tumors were injected i.v. withPEGylated trimer H-NOX (L144F) at 650 mg/kg when tumor volume reached˜300-350 mm³ (˜10-14 days after tumor cell subcutaneous implantation).Prior to euthanasia, mice were injected with the exogenous hypoxiamarker pimonidazole at 60 mg/kg and tumors were harvested. Pimonidazole(FIG. 2A) (Hypoxyprobe-1) and HIF-1α (FIG. 2B) levels were measured bycompetitive (pimonidazole) and sandwich (HIF-1α, Abcam) ELISAs,respectively. Graphs show quantification of pimonidazole and HIF-1αsignals after PEGylated H-NOX (L144F) administration. Vehicle, 1h and4h: n=22, 7h: n=18, 12h: n=16, 24h: n=6. Results from 4 independentexperiments. Mean values+/−SEM. ****p<0.0001, ***p<0.001, **p<0.01,*p<0.05 by one-way ANOVA and Bonferroni's post-hoc tests. (FIG. 2C)Tumors were assessed for the accumulation of PEGylated H-NOX (L144F) bysandwich H-NOX ELISA at 1, 4, 7, 16 and 24 hours after injection andresults expressed per gram of tumor tissue. Seven to eight week oldNu/Nu female mice were subcutaneously implanted with 3×10⁶ of H460 humanlung cancer cells and monitored until the tumors reached average size of˜300 mm³ (10-14 days post-implantation of tumor cells). Mice bearing200-350 mm³ xenograft tumors were injected i.v. with bolus vehicle(formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4 mM EDTA, and 10 mMreduced glutathione at pH 7) or formulation buffer containing 650 mg/kgof PEGylated trimer H-NOX (L144F). To measure tumor hypoxia, prior toeuthanasia, mice were injected with the exogenous hypoxia markerpimonidazole at 60 mg/kg. Tumors were harvested and homogenized in anextraction buffer (Abcam kit # ab117996) supplemented withanti-proteases. Protein concentration was quantified in each tumor usinga Bradford assay. Samples were assayed for pimonidazole (Hypoxyprobe-1)amount using a competitive ELISA assay developed by Omniox and forHIF-1α using the Abcam ELISA kit (ab117996).

In H460 lung carcinoma mouse model maximum oxygenation was achievedbetween 4h and 8h and it correlated with the peak of H-NOX (L144F) tumoraccumulation as assessed by ELISA (FIG. 2C).

For assessment of the H-NOX (L144F) tumor accumulation, tumors wereharvested at different timepoints after injection. Tumors werehomogenized in an extraction buffer (Abcam kit # ab117996) supplementedwith anti-proteases and protein concentration was quantified in eachtumor using a Bradford assay. PEGylated H-NOX (L144F) concentration wasquantified by a sandwich ELISA for H-NOX developed by Omniox andnormalized to tumor weight.

While supplemental oxygenation of animals successfully increasedoxygenation of mouse tumor tissue at 5-10 mmHg oxygen concentration, ithad no effect on regions with lower oxygen levels (<5 mmHg). Bycontrast, PEGylated trimer Tt H-NOX L144F is capable of increasingoxygenation even in severely hypoxic tumor tissue (<5 mmHg). This islikely due to PEGylated trimer Tt H-NOX L144F's superior tissuepenetration that enables oxygen delivery to areas beyond oxygen gradientdiffusion limits. Moreover, while maximum supplemental oxygenation ofmouse tumors is achieved with exposing animals continuously to 95%-100%breathing oxygen [increasing risk of hyperoxic and inflammatory damageto the normal tissues (Kallet & Matthay, 2013 Respir Care,58(1):123-141; Thiel et al., 2005 PLoS Biol, 3(6), e174)], single bolusi.v. dose of PEGylated trimer TL H-NOX L144F can maintain tissueoxygenation for more than 7 hours without increasing oxygen levels innormal tissues. A control Tt H-NOX protein (wild type variant)—that isnot capable of releasing oxygen at oxygen concentrations present inhypoxic tissues-did not have any effect on tumor oxygenation (FIG. 3C).

Seven to eight week old Nu/Nu female mice were subcutaneously implantedwith 3×10⁶ of H460 human lung cancer cells and monitored until thetumors reached average size of ˜500 nm³(10-18 days post-implantation oftumor cells). Mice bearing H460 tumors were anesthetized with isofluranemixed in 20% of oxygen and the OxyLite™ probe (Oxford Optronix, UK) wasimplanted into H460 subcutaneous xenograft tumors using amicromanipulator. The OxyLite™ consists of the ruthenium chloride dyeheld in a polymer matrix of 230 μm in diameter at the tip. Afterequilibration for ˜20-30 minutes, pO₂ was measured using opticalfluorescence sensors attached to a four-channel unit. A low starting pO₂confirmed entry into hypoxic tissue away from neighbouring blood vessels(˜0.2 mmHg; except in FIG. 3D where 5 mmHg). After probe implantation,probe was left for ˜20-30 min in order for pO₂ measurements tostabilize, and mice were given to respire 100% O₂ (FIG. 3B, FIG. 3D) orwere injected with PEGylated H-NOX (L144F in FIG. 3A, wt in FIG. 3C) andfluorescent quenching was recorded.

The superior ability of PEGylated trimer Tt H-NOX L144F to deliveroxygen to hypoxic tumor regions relative to the administration of thehyperoxic gas was further demonstrated by more efficient radiation tumorcell kill (FIG. 4).

Mice bearing H460 subcutaneous xenograft tumors (200-350 mm³) weretreated with 10 Gy alone or in combination with 650 mg/kg of PEGylatedtrimer H-NOX (L144F) injected i.v. 7 hours prior to irradiation. Tumorswere extracted after irradiation and processed for clonogenic assay.Cell numbers were counted 10-14 days later in triplicate samples fromeach tumor. Each dot on the graph represents average surviving fractionfor one tumor. Mean values+/−SEM (n=3 per experiment). Seven to eightweek old Nu/Nu female mice were subcutaneously implanted with 3×10⁶ ofH460 human lung cancer cells and monitored until the tumors reachedaverage size of ˜300 mm³ (10-14 days post-implantation of tumor cells).Mice bearing 200-350 mm³ xenograft tumors were irradiated with 10 Gyalone or in combination with intravenous delivery of either a bolusvehicle (formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4 mM EDTA,and 10 mM reduced glutathione at pH 7) or a formulation buffercontaining 650 mg/kg of PEGylated H-NOX (L144F). Mice were sacrificedafter irradiation and tumors were harvested and processed for an ex-vivoclonogenic assay. Briefly, tumors cells were minced into fine pieceswith a scalpel and digested for ˜30 minutes with an enzymatic cocktailcontaining a mix of collagenase (200 U/ml), hyaluronidase (200 U/ml) andDNAse (10 ml of cocktail/g of tumor). Extracted cells were then countedand seeded at 500/125/25 cells per well for untreated and 2000/500/100cells per well for irradiated tumor samples in a 6 well plate induplicate. After 10-12 days, cell colonies were fixed with PFA andstained with crystal violet. The number of clones (over 50 cells) wascounted and the plating efficiency was calculated in untreated samples(number of cells counted in well÷number of cells plated x 100).Surviving fraction was calculated in all samples (number of cellscounted÷plating efficiency x 100).

Following PEGylated trimer Tt H-NOX L144F administration, therewas >15-fold increase in radiation treatment efficacy in all PEGylatedtrimer Tt H-NOX L144F-treated tumors that reduced the surviving fractionof tumor cells from 30% in the 10 Gy treatment alone group to <2% in theH-NOX (L144F)-pretreated tumor. In the same experiment, treatment ofmice bearing tumors with 100% oxygen showed variable increase inradiation enhancement (˜3 fold) probably resulting from unequal tumoroxygenation between individual tumors likely due to uneven vasculardensity between tumors.

Example 2. H-NOX Acts as an Immunoactivator Enhancing Host Anti-TumorResponses

PEGylated trimer Tt H-NOX L144F-induced oxygenation inhibits the HIF-1αpathway (FIG. 2B) and relieves HIF-1α-dependent and HIF-1α-independenttumor immunosuppression. Mice bearing H460 subcutaneous xenograft tumors(200-350 mm³) were either treated with vehicle alone or with PEGylatedtrimer H-NOX (L144F) and harvested 7, 16 or 24 hours after injection forqRT-PCR analysis. Mean values+/−SEM. N=5-6 per group, *p<0.05 by t-test.Treatment with a single dose of H-NOX resulted in significantdownregulation of HIF-1α and its effectors including direct and indirectmodulators of the host's immune response: PD/PDL-1 and VEGF signaling,metabolic and growth factor regulators (FIG. 5).

Seven to eight week old Nu/Nu female mice were subcutaneously implantedwith 3×10⁶ of H460 human lung cancer cells and monitored until thetumors reached average size of ˜300 mm³ (10-14 days post-implantation oftumor cells). Mice bearing 200-350 mm³ xenograft tumors were injectedwith either a bolus vehicle (formulation buffer: 50 mM succinate, 50 mMNaCl, 3.4 mM EDTA, and 10 mM reduced glutathione at pH 7) or aformulation buffer containing 650 mg/kg of PEGylated trimer H-NOX(L144F). To prepare samples for qRT-PCR analysis, mice were sacrificedand tumors excised. Total RNA was extracted from tumor samples using theRNeasy kit (QIAGEN) according to the manufacturer instructions. Reversetranscription and real-time PCR (RT-PCR) on a StepOnePlus™ Real-Time PCRSystem (Applied Biosystems) were performed as described. 25 μL reactionwas prepared using 2 μL of cDNA template, 12.5 μL of SYBR® Green PCRMaster Mix (Applied Biosystems) and 1 μL of the following sense andantisense primers: VEGF: forward, 5′-CAATCGAGACCCTGGTGGA-3′ (SEQ IDNO:23); reverse, 5′-GCACACACTCCAGGCCCT-3′ (SEQ ID NO:24); Glut1:forward, 5′-CAACCAGACATGGGTCCAC-3′ (SEQ ID NO:25); reverse,5′-GTTAACGAAAAGGCCCACAGA-3′ (SEQ ID NO:26); PDL1: forward,5′-GTTGTGGATCCAGTCACCTCT-3′ (SEQ ID NO:27); reverse,5′-GATTCTCAGTGTGCTGGTCAC-3′(SEQ ID NO:28); L7: forward,5′-CAAGGAGGAAGCTTATCTATGAA-3′(SEQ ID NO:29); reverse,5′-ATTTGACGAAGGCGAAGAAGCT-3′ (SEQ ID NO:30). Thermocycling conditionswere as follows: initial step was 10 min at 95° C., then 40 cycles of 15s denaturation at 95° C. followed by 1 min annealing and extension at60° C. Results were analyzed with the StepOne Software v2.0 using thecomparative CT method. Transcripts of gene of interest were normalizedagainst the transcripts of the mouse ribosomal protein L7 housekeepinggene, and were presented as fold change relative to the L7 transcriptcontent.

For example, PEGylated trimer Tt H-NOX L144F-mediated downregulation ofthe HIF-1α and A2AR adenosinergic signaling may result in activation andrecruitment of effector T cells to the tumor tissue leading to increasedlymphocyte tumor infiltration, decrease in metastatic tumor growth andtumor regression. Furthermore, H-NOX-induced oxygenation of tumors mayreduce immunoevasion of tumor cells by inhibiting multiplehypoxia-dependent mechanisms including downregulation of MHC1 andupregulation of PDL-1 expression on tumor cell surface, and activationand recruitment of myeloid-derived suppressor cells (MDSC) includingTAMs that directly suppress immune effector cells as well as promoteangiogenesis and metastasis (see FIG. 1B). H-NOX treatment may inhibitrecruitment of macrophages to TME by downregulating VEGF, CSF1 and otherHIF-1-dependent cytokine signaling (Chaturvedi et al., 2014 Proc NatlAcad Sci USA, 111(20):E2120-2129; Lewis & Hughes, 2007 Breast CancerRes, 9(3):209) as well as HIF-1- and HIF-2-mediated macrophageactivation (Fang et al., 2009 Blood, 114(4):844-859; Takeda et al., 2010Genes Dev, 24(5):491-501).

Finally, in stimulating host's anti-tumor immune response, H-NOX may actas co-activator enhancing other targeted cancer immunotherapies such as,but not limited to, anti-PD1 (programmed cell death protein 1),anti-PDL-1 (programmed cell death protein ligand 1), anti-CTLA-4 ortherapies targeting other immune checkpoints' regulators, anti-cancervaccines, adoptive immune cell therapies or combinations thereof. Forexample, H-NOX may be administered to cancer patients prior to and inconjunction with dual PD1/CTLA-4 blockade therapy or in combination withPDL-1 treatment in patients with PDL1+ tumors. It may also act as anadjuvant to other cancer treatments including, but not limited to,chemotherapy, radiation therapy or other non-immune targeted orcell-based therapies that may benefit from active anti-tumor immunedefenses. Indeed, H-NOX may synergize with radiation by simultaneouslystimulating anti-tumor immune response towards radiation exposedtumor-specific antigens from damaged tumor tissue (Demaria et al., 2005Int J Radiat Oncol Biol Phys, 63(3), 655-666) and oxygen-dependent tumorcell killing (Brown, 2010 Int J Radiat Biol, 86(11), 907-917). Duringradiotherapy, H-NOX may also act as normal tissue radioprotectant byameliorating hypoxia resulting from radiation-induced vascular damage.

Example 3. Measurement of Hypoxia and T Cells in B16F10 and CT26Subcutaneous Tumors and Intracranial GL261-Luciferase Tumors

Generation of B16F10 and CT26 Subcutaneous Tumors and IntracranialGL261-Luciferase Tumors.

Six to eight week old C57BL/6J female mice were subcutaneously implantedwith 1×10⁶ B16F10 mouse melanoma cancer cells on the flank (FIG. 7A).Six to eight week old BALB/c female mice were subcutaneously implantedwith 1×10⁶ CT26 colon tumor cells on the flank (FIG. 7B). Male C57BL/6Jweighting 20 g were injected with 3×10⁵ GL261-luc cells intracraniallyinto the right caudate nucleus (+0.5 mm A/P, +2.3 mm M/L and −3.2 mmD/V) (FIG. 7C). Intracranial tumors were allowed to grow for 21 daysbefore sacrifice. Subcutaneous tumors were measured 3 times a week usingcalipers and tumor size was calculated based on the formula: (length xwidth²)+0.5. Once the tumors reached an average size of −300 mm³ (10-14days post-implantation of tumor cells), treatment was initiated.

Treatment.

Mice bearing 200-400 mm³ subcutaneous tumors or day 21 intracranialtumors were injected with the exogenous hypoxia marker pimonidazole ip.(60 mg/kg, Hypoxyprobe, Burlington Mass.) 1-8 hours prior to sacrifice.

Immunohistochemistry.

Rodents were euthanized and tumors resected for immunohistochemistry(IHC) assay. Tumors were frozen in OCT and sectioned at 12 μM for IHCprocessing. Sections were fixed with 4% PFA for 15 minutes at 4° C.,then blocked and permeabilized with 5% BSA, 5% goat serum, and 0.1%Tween 20 for 1-2 hours at room temperature. Sections were then incubatedwith rabbit anti-pimonidazole (Hypoxyprobe, 1:100) (FIGS. 7A, 7B, 7C,top panels) and rat anti-CD3, rat anti-CD4 (Biolegend, 1:50) or ratanti-CD8 (Biolegend, 1:50) antibodies overnight at 4° C. (FIGS. 7A, 7B,7C, middle panels), followed by anti-rabbit or anti-rat secondaryantibodies (1:1000, Jackson Immunoresearch Laboratories, West Grove,Pa., USA) for 2 hours at room temperature. The sections were mounted inSlowFade DAPI (Invitrogen). Sections were imaged with an HD AxiolmagerZeiss microscope equipped with a CCD digital camera.

Quantification.

In each animal, the number of CD3+, CD4+ or CD8+ T cells was counted inpimonidazole-positive and pimonidazole-negative areas in 2-4 picturesper section in 5 tumor sections spanning 1-1.5 mm of the tumorthickness. The sum of CD4+ and CD8+ cells in each area was divided bythe sum of pimonidazole-positive and pimonidazole-negative areas toobtain the total number of T cells per mm² of tumor tissue (FIGS. 7A,7B, and 7C, bottom panels). Hypoxic regions of tumors (H) showed 2.5 toover 10-fold less T cells than normoxic regions (N).

Example 4. H-NOX Treatment of Hypoxic Tumors

Generation of B16F10 Subcutaneous Tumors.

Six to eight week old C57BL/6J female mice were subcutaneously implantedwith 1×10⁶ of B16F10 mouse melanoma cancer cells on the flank. Tumorswere measured 3 times a week using calipers and tumor size wascalculated based on the formula: (length x width²)÷0.5. Once the tumorsreached an average size of ˜300 mm³ (10-14 days post-implantation oftumor cells), treatment was initiated.

Treatment.

Mice bearing 200-400 mm³ tumors were randomized in each treatment groupbased on tumor size and injected intratumorally with vehicle(formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4 mM EDTA, and 10 mMreduced glutathione at pH 7) or 100 μl of formulation buffer containing2 mg of PEGylated H-NOX (L144F). One hour prior to vehicle or H-NOXtreatment, mice were injected with the exogenous hypoxia markerpimonidazole ip. (60 mg/kg, Hypoxyprobe, Burlington Mass.).

Immunohistochemistry.

6 hours after H-NOX injection, rodents were euthanized and tumorsresected for immunohistochemistry (IHC) assay. Tumors were frozen in OCTand sectioned at 12 μM for IHC processing. Sections were fixed with 4%PFA for 15 minutes at 4° C., then blocked and permeabilized with 5% BSA,5% goat serum, and 0.1% Tween 20 for 1-2 hours at room temperature.Sections were then incubated with rabbit anti-pimonidazole (Hypoxyprobe,1:100) or rabbit anti-carbonic anhydrase IX (CAIX, Novus Biological1:1000) and rat anti-CD4 (Biolegend, 1:50) or rat anti-CD8 (Biolegend,1:50) antibodies overnight at 4° C., followed by anti-rabbit or anti-ratsecondary antibodies (1:1000, Jackson Immunoresearch Laboratories, WestGrove, Pa., USA) for 2 hours at room temperature. The sections weremounted in SlowFade DAPI (Invitrogen). Sections were imaged with an HDAxiolmager Zeiss microscope equipped with a CCD digital camera (FIGS. 8and 9B).

Quantification.

In each animal, the number of CD4+ and CD8+ T cells was counted inpimonidazole-positive, pimonidazole-negative, CAIX-positive andCAIX-negative areas in 4 pictures per section in 5 tumor sectionsspanning 1-1.5 mm of the tumor thickness. The sum of CD4+ and CD8+ cellsin each area was divided by the sum of pimonidazole-positive,pimonidazole-negative, CAIX-positive and CAIX-negative areas to obtainthe total number of T cell per mm² of tumor tissue. Results shown inFIGS. 8 and 9A demonstrate that OMX treatment as compared to the vehiclecontrol (formulation buffer) enhances accumulation of CD4+ and CD8+lymphocytes in previously pimondazole-negative (FIG. 8) or CAIX-negative(FIG. 9B) labeled hypoxic regions of the tumors.

Example 5. Measurement of Tumor Hypoxia and Tumor Vessels

Generation of H460, B16F10 and CT26 Subcutaneous Tumors and IntracranialGL261-Luciferase Tumors.

Seven to eight week old Nu/Nu female mice were subcutaneously implantedwith 3×10⁶ of H460 human lung cancer cells in the hind limb. Six toeight week old C57BL/6J female mice were subcutaneously implanted with1×10⁶ B16F10 mouse melanoma cancer cells on the flank. Six to eight weekold BALB/c female mice were subcutaneously implanted with 1×10⁶ CT26colon tumor cells on the flank. Male C57BL/6J weighting 20 g wereinjected with 3×10⁵ GL261-luc cells intracranially into the rightcaudate nucleus (+0.5 mm A/P, +2.3 mm M/L and −3.2 mm D/V). Intracranialtumors were allowed to grow for 21 days before sacrifice. Subcutaneoustumors were measured 3 times a week using calipers and tumor size wascalculated based on the formula: (length x width²⁾÷0.5. Once the tumorsreached an average size of ˜300 mm³ (10-14 days post-implantation oftumor cells), treatment was initiated. Treatment. Mice bearing 200-400mm³ subcutaneous tumors or day 21 intracranial tumors were injected withthe exogenous hypoxia marker pimonidazole ip. (60 mg/kg, Hypoxyprobe,Burlington Mass.) 1-8 hours prior to sacrifice.

Immunohistochemistry and ELISA.

Rodents were euthanized and tumors resected for immunohistochemistry(IHC) and ELISA assays. For ELISA, B16F10, CT26 and H460 tumors werehomogenized in an extraction buffer (Abcam kit # ab117996) supplementedwith anti-proteases. Protein concentration was quantified in each tumorusing a Bradford assay and samples were assayed for hypoxia levels usinga competitive Pimonidazole (Hypoxyprobe-1, Hypoxyprobe, BurlingtonMass.) ELISA assay. For IHC, GL261 tumors were frozen in OCT andsectioned at 12 μM for IHC processing. Sections were fixed with 100%methanol for 20 minutes at −20° C., then blocked and permeabilized with5% BSA, 5% goat serum, and 0.1% Tween 20 for 1-2 hours at roomtemperature. Sections were then incubated with rabbit anti-pimonidazole(Hypoxyprobe, 1:100) and rat anti-CD31 (BD Bioscience, 1:50) antibodiesovernight at 4° C., followed by anti-rabbit or anti-rat secondaryantibodies (1:1000, Jackson Immunoresearch Laboratories, West Grove,Pa., USA) for 2 hours at room temperature. The sections were mounted inSlowFade DAPI (Invitrogen). Sections were imaged with an HD AxiolmagerZeiss microscope equipped with a CCD digital camera (FIG. 10).

Quantification.

For the pimonidazole ELISA, quantification of the IC₅₀ values (“Kd”)were performed with a 5-parameter fit of the standard curve and valueswere normalized according to the protein concentration in each sample.For GL261 IHC, in each animal, the percent of pimonidazole+ area withinthe tumor tissue was determined using ImageJ (1-2 pictures per sectionin 5 tumor sections spanning 1 mm of the tumor thickness) (FIG. 10).These data show that while there is a range in the levels of hypoxiabetween individual animals and between tumor types, its presence issignificant in a majority of the tumors of the examined sizes.

Example 6. Measurement of H-NOX Accumulation in Tumors

Generation of B16F10 and CT26 Subcutaneous Tumors and IntracranialGL261-Luciferase Tumors.

Six to eight week old C57BL/6J female mice were subcutaneously implantedwith 1×10⁶ B16F10 mouse melanoma cancer cells on the flank. Six to eightweek old BALB/c female mice were subcutaneously implanted with 1×10⁶CT26 colon tumor cells on the flank. Male C57BL/6J weighting 20 g wereinjected with 3×10⁵ GL261-luc cells intracranially into the rightcaudate nucleus (+0.5 mm A/P, +2.3 mm M/L and −3.2 mm D/V). Intracranialtumors were allowed to grow for 21 days before sacrifice. Subcutaneoustumors were measured 3 times a week using calipers and tumor size wascalculated based on the formula: (length x width²)+0.5. Once the tumorsreached an average size of −300 mm³ (10-14 days post-implantation oftumor cells), treatment was initiated.

Treatment. Mice bearing 200-400 mm³ subcutaneous tumors were randomizedin each treatment group based on tumor size and injected intravenously(650 mg/kg), subcutaneously (650 mg/kg), or intratumorally (2 mg, 100μl) with vehicle (formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4mM EDTA, and 10 mM reduced glutathione at pH 7) or formulation buffercontaining PEGylated H-NOX (L144F). Mice bearing day 21 intracranialtumors were randomized in each treatment group based on bioluminescentsignal measured with the Xenogen IVIS spectrum and injectedintravenously with formulation buffer alone or containing 750 mg/kg ofH-NOX (L144F).

Measurement of PEGylated H-NOX (L144F) Accumulation in SubcutaneousTumor Tissue.

Tumors were harvested 6h (B16F10) or 8h (CT26) after H-NOX or vehicleinjection. Tumors were homogenized in an extraction buffer (Abcam kit #ab117996) supplemented with anti-proteases and protein concentration wasquantified in each tumor using a Bradford assay. PEGylated H-NOX (L144F)concentration was quantified by a sandwich ELISA ELISA (detectionsensitivity at 1 ng/ml) for H-NOX and normalized to tumor weight toexpress H-NOX amount in μg/g tumor tissue. Quantification of H-NOXlevels in tumor lysates was determined by 5-parameter fit of thestandard curve.

Biodistribution of H-NOX (L144F) in GL261 by IHC.

2h after H-NOX injection, rodents were euthanized and tumors resectedfor immunohistochemistry (IHC) assay. Tumors were frozen in OCT andsectioned at 12 μM for IHC processing. Sections were fixed with 100%methanol for 20 minutes at −20° C., then blocked and permeabilized with5% BSA, 5% goat serum, and 0.1% Tween 20 for 1-2 hours at roomtemperature. Sections were then incubated with rabbit anti-H-NOX (1:500,custom-made rabbit polyclonal produced by AnaSpec Inc, Fremont, Calif.)and rat anti-CD31 (BD Bioscience, 1:50) antibodies overnight at 4° C.,followed by anti-rabbit or anti-rat secondary antibodies (1:1000,Jackson Immunoresearch Laboratories, West Grove, Pa., USA) for 2 hoursat room temperature. The sections were mounted in SlowFade DAPI(Invitrogen). Sections were imaged with an HD Axiolmager Zeissmicroscope equipped with a CCD digital camera. In each animal, thepercent of H-NOX-positive area within the tumor tissue was determinedusing ImageJ (1-2 pictures per section in 5 tumor sections spanning 1 mmof the tumor thickness).

Example 7. Measurement of Hypoxia and T Cells in Canine Oral MelanomaTumors

Canine Oral Melanoma.

Pet dogs with oral melanoma tumors were recruited for the study withowners' consent and injected intravenously (slow infusion) withPEGylated H-NOX (L144F) 4h prior to surgery. Tissue extracted fromsurgery was analyzed by IHC.

Immunohistochemistry.

4 hours after H-NOX injection, tumors were resected, frozen in OCT andsectioned at 12 μM for IHC processing. Sections were fixed with 4% PFAfor 15 minutes at 4° C., then blocked and permeabilized with 5% BSA, 5%goat serum, and 0.1% Tween 20 for 1-2 hours at room temperature.Sections were then incubated with rabbit anti-carbonic anhydrase IX(CAIX, Novus Biological 1:1000) or rabbit anti-H-NOX (1:500, custom-maderabbit polyclonal produced by AnaSpec Inc, Fremont, Calif.) and ratanti-CD4 (Abd Serotech, 1:50) or rat anti-CD8 (Abd Serotech, 1:50)antibodies overnight at 4° C., followed by anti-rabbit or anti-ratsecondary antibodies (1:1000, Jackson Immunoresearch Laboratories, WestGrove, Pa., USA) for 2 hours at room temperature. The sections weremounted in SlowFade DAPI (Invitrogen). Sections were imaged with an HDAxiolmager Zeiss microscope equipped with a CCD digital camera (FIG.11). Images revealed presence of high lymphocyte numbers in tumorregions that expressed hypoxia marker CAIX indicative of hypoxic stateprior to OMX administration suggesting that OMX treatment relievedimmunosuppressive microenvironment and allowed lymphocyte infiltration.

Example 8. Correlation of Tumor Volume, Tumor Hypoxia and Reduced T CellInfiltration

4T1-Luc Tumor Model.

8 week-old female BALB/c mice were purchased from Charles River Labs.Luciferase-expressing 4T1 mouse breast tumor cells (4T1-Fluc-Neo; ImanisLife Sciences) were grown in RPMI medium supplemented with 10% fetalbovine serum, 1× penicilin/streptomycin, and 0.7 mg/ml G-418(InvivoGen). Cells were trypsinized and resuspended in a 50:50 mixtureof medium:Matrigel (Corning), and 2×10⁵ cells in 100 μl volume wasinjected subcutaneously into mice. At day 10 and day 14post-implantation, tumors were measured and volumes calculated (length xwidth x height x 0.523), mice were injected simultaneously with 120mg/kg pimonidazole i.p. (PIMO, Hypoxyprobe) and 30 mg/kg EF5 i.v.(Hypoxia Imaging Center), sacrificed 90 min post-PIMO/EF5 injection, andtumors were harvested. Harvested tumors were frozen in OCT forimmunostaining, as well as dissociated into single cells using agentleMACS dissociator followed by incubation with 0.75 mg/mlcollagenase/dispase (Roche) at 37° C. with shaking for 45 min.Dissociated cells were passed through 70 μm filters.

Flow Cytometry.

Unfixed dissociated cells were stained with antibodies for T cells(hamster anti-mouse CD3-AlexaFluor 488, clone 145-2C11, eBioscience; ratanti-mouse CD4-APC, clone RM4-5, BD Biosciences; rat anti-mouse CD8-PE,clone 53-6.7, BD Biosciences), and flow cytometry was performed using aFACSCalibur. Spleens were used as T cell positive controls for gatingpurposes. Also after filtration of dissociated cells through 70 μmfilters, cells were stained with viability dye 570 (BD Biosciences),fixed with formalin and methanol, stained with antibodies for hypoxiamarkers (rabbit anti-pimonidazole, Hypoxyprobe, followed by donkeyanti-rabbit AlexaFluor 647; mouse anti-EF5 conjugated to AlexaFluor 488,Hypoxia Imaging Center), and analyzed on a FACSCalibur. Flow cytometrydata were analyzed using FlowJo.

Immunofluorescence Staining.

Frozen sections were cut at 10 μm, fixed with 4% PFA, stained withprimary antibodies (rat anti-mouse CD4, rat anti-mouse CD8, rabbitanti-PIMO), followed by secondary antibodies (donkey anti-rat AlexaFluor594, donkey anti-PIMO AlexaFluor 488), and counterstained with DAPI.

As shown in FIGS. 13A-13K, larger tumor size correlates with enhancedhypoxia and reduced lymphocyte infiltration in subcutaneous 4T1-Lucsyngeneic tumors. FIG. 12A shows tumor volumes on day 10 and day 14post-implantation. FIG. 12B shows the fraction of lymphocytes within theviable cell population and FIG. 12C shows the absolute lymphocyte cellnumbers within the viable population. Negative correlations betweentumor volume and percentage lymphocytes (FIG. 12D) and betweenpercentage hypoxia and percentage lymphocytes (FIG. 12F) weredemonstrated whereas the relationship between tumor volume andpercentage hypoxia showed a positive correlation (FIG. 12E). Negativecorrelations were also seen between tumor volume and percentageCD3-positive T cells (FIG. 12G), between tumor volume and percentageCD4-positive T cells (FIG. 12H), between tumor volume and percentageCD8-positive T cells (FIG. 12I), between tumor volume and percentageCD3-CD4-double-positive T cells (FIG. 12J), and between tumor volume andpercentage CD3-CD8-double-positive T cells (FIG. 12K).

FIGS. 13A-13F show that hypoxic tumor regions are immunosuppressive andexhibit reduced T cell infiltration in subcutaneous 4T1-Luc syngeneicmouse tumors.

SEQUENCES Tt WTATGAAGGGGACAATCGTCGGGACATGGATAAAGACCCTGAGGGACCTTTACGGGAATGATGTGGTTGATGAATCTTTAAAAAGTGTGGGTTGGGAACCAGATAGGGTAATTACACCTCTGGAGGATATTGATGACGATGAGGTTAGGAGAATTTTTGCTAAGGTGAGTGAAAAAACTGGTAAAAATGTCAACGAAATATGGAGAGAGGTAGGAAGGCAGAACATAAAAACTTTCAGCGAATGGTTTCCCTCCTATTTTGCAGGGAGAAGGCTAGTGAATTTTTTAATGATGATGGATGAGGTACACCTACAGCTTACCAAGATGATAAAAGGAGCCACTCCTCCAAGGCTTATTGCAAAGCCTGTTGCAAAAGATGCCATTGAAATGGAGTACGTTTCTAAAAGAAAGATGTACGATTACTTTTTAGGGCTTATAGAGGGTAGTTCTAAATTTTTCAAGGAAGAAATTTCAGTGGAAGAGGTCGAAAGAGGCGAAAAAGATGGCTTTTCAAGGCTAAAAGTCAGGATAAAATTTAAAAACCCCGTITTTGAGTGA (SEQ ID NO: 1)MKGTIVGTWIKTLRDLYGNDVVDESLKSVGWEPDRVITPLEDIDDDEVRRIFAKVSEKTGKNVNEIWREVGRQNIKTFSEWFPSYFAGRRLVNFLMMMDEVHLQLTKMIKGATPPRLIAKPVAKDAIEMEYVSKRKMYDYFLGLIEGSSKFFKEEISVEEVERGEKDGFSRLKVRIKFKNPVFE (SEQ ID NO: 2) foldon domainGGTTATATTCCTGAAGCTCCAAGAGATGGGCAAGCTTACGTTCGTAAAGATGGCGAATGGGTATTACTTTCTACCTTTTTA (SEQ ID NO: 3) GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 4) L2 WTATGATGTCTATGAAAGGAATCATATTCAACGAATTTCTCAATTTTGTAGAAAAAAGTGAATCCTACACCCTGGTAGATCAAATTATTATGGATAGTCATTTGAAGTCCCATGGTGCCTACACGTCTATCGGTACATACTCTCCCAAAGAATTATTTCAATTGGTTAAAGCGCTTGCTATGAAAAATGGCAAACCAACATCAGTGATTTTACAAGAATATGGTGAGTATTTGTTTGAGGTTTTTGCAAAAAAATATCCTCAATTTTTCAGGGAAAAAAAGTCGGTGTTTCAATTTTTGGAAGCGCTTGAAACACATATTCATTTCGAAGTGAAAAAATTGTATGACTATACTGAACTACCCCATTTTGAATGCCAATATCACAGTCAAAATCAAATGGAAATGATTTACACTTCTTCGCGTCCTTTGGCCGATTTTGCGGAAGGTTTAATAAAAGGTTGTATTAAATATCATAAAGAAAACATGACTATTGTTCGTGAAAATCTGCCTGCAAAAACAGGCTTTAAGGTAAGATTTGTATTAACAAAAGGCGATCCTGATGAGTGA (SEQ ID NO: 9)MMSMKGIIFNEFLNFVEKSESYTLVDQIIMDSHLKSHGAYTSIGTYSPKELFQLVKALAMKNGKPTSVILQEYGEYLFEVFAKKYPQFFREKKSVFQFLEALETHIHFEVKKLYDYTELPHFECQYHSQNQMEMIYTSSRPLADFAEGLIKGCIKYHKENMTIVRENLPAKTGFKVRFVLTKGDPDE (SEQ ID NO: 10) L1 WTATGAAAGGTATCGTTTTTACCTCCTTAAATGACATGATTATAGAACAATTTGGCATAGAAACCTGGGACCAACTCGTATCCTCACTAGACCTTCCAAGTGGTGGAAGTTATACAGCAGGCGGCACTTACTOGGATACAGAATTTCAGCAATTGATTAAGGCCATTGCGAAGAGGACCAATCAGCACGCTTCTGTTTTTTTAGAGGCCTTTGGTGAATACATGTTTCCTATCTTATCGAGTAAGTGCGCAATTTTTTTAAAAAAGGACATGACATTAAAAGAATTTTTAAAAAGCATTGATGGAACAATTCATGTGGAAGTAGAAAAGTTATACCCAGATGAAACATTACCTACCATTAGCTATGAAGAGCCTGCTGCAAACCAATTGGTTATGGTGTATCGATCGCATAGAAGACTCTGTCATTTTGCAATGGGGCTCATCCAGGGAGCAGCGCAACATTTTAAAAAGAAAATTACCATTAAGCAGACTCACTGCATGTTAAAAAAAGATGATCATTGTCGTTTGGAGATTACCTTTGAGTGA (SEQ ID NO: 11)MKGIVFTSLNDMIIEQFGIETWDQLVSSLDLPSGGSYTAGGTYSDTEFQQLIKAIAKRTNQHASVFLEAFGEYMFPILSSKCAIFLKKDMTLKEFLKSIDGTIHVEVEKLYPDETLPTISYEEPAANQLVMVYRSHRRLCHFAMGLIQGAAQHFKKKITIKQTHCMLKKDDHCRLEITFE (SEQ ID NO: 12) Homo sapiens WT (1-385)ATGTACGGATTTGTGAATCACGCCCTGGAGTTGCTGGTGATCCGCAATTACGGCCCCGAGGTGTGGGAAGACATCAAAAAAGAGGCACAGTTAGATGAAGAAGGACAGTTTCTTGTCAGAATAATATATGATGACTCCAAAACTTATGATTTGGTTGCTGCTGCAAGCAAAGTCCTCAATCTCAATGCTGGAGAAATCCTCCAAATGTTTGGGAAGATGTTTTTCGTCTTTTGCCAAGAATCTGGTTATGATACAATCTTGCGTGTCCTGGGCTCTAATGTCAGAGAATTTCTACAGAACCTTGATGCTCTGCACGACCACCTTGCTACCATCTACCCAGGAATGCGTGCACCTTCCTTTAGGTGCACTGATGCAGAAAAGGGCAAAGGACTCATTTTGCACTACTACTCAGAGAGAGAAGGACTTCAGGATATTGTCATTGGAATCATCAAAACAGTGGCACAACAAATCCATGGCACTGAAATAGACATGAAGGTTATTCAGCAAAGAAATGAAGAATGTGATCATACTCAATTTTTAATTGAAGAAAAAGAGTCAAAAGAAGAGGATTTTTATGAAGATCTTGACAGATTTGAAGAAAATGGTACCCAGGAATCACGCATCAGCCCATATACATTCTGCAAAGCTTTTCCTTTTCATATAATATTTGACCGGGACCTAGTGGTCACTCAGTGTGGCAATGCTATATACAGAGTTCTCCCCCAGCTCCAGCCTGGGAATTGCAGCCTTCTGTCTGTCTTCTCGCTGGTTCGTCCTCATATTGATATTAGTTTCCATGGGATCCTTTCTCACATCAATACTGTTTTTGTATTGAGAAGCAAGGAAGGATTGTTGGATGTGGAGAAATTAGAATGTGAGGATGAACTGACTGGGACTGAGATCAGCTGCTTACGTCTCAAGGGTCAAATGATCTACTTACCTGAAGCAGATAGCATACTTTTTCTATGTTCACCAAGTGTCATGAACCTGGACGATTTGACAAGGAGAGGGCTGTATCTAAGTGACATCCCTCTGCATGATGCCACGCGCGATCTTGTTCTTTTGGGAGAACAATTTAGAGAGGAATACAAACTCACCCAAGAACTGGAAATCCTCACTGACAGGCTACAGCTCACGTTAAGAGCCCTGGAAGATTGA (SEQ ID NO: 13)MYGFVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMFFVFCQESGYDTILRVLGSNVREFLQNLDALHDHLATIYPGMRA-SFRCIDAEKGKGLILHYYSEREGLQDIVIGIIKTVAQQIHGTEIDMKVIQQRNEECDHTQFLIEEKESKEEDFYEDLDRFEENGTOESRISPYTFCKAFPFHIIFDRDLVVTQCGNAIYRVLPQLQPGNCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDELTGTEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRAGLYLSDIPLHDATADLVLLGEQFREEYKLIQELEILTDRLOLTLRALED (SEQ ID NO: 14) Homo sapiens β2 (1-217)ATGTATGGATTCATCAACACCTGCCTGCAGTCTCTTGTGACAGAGAAATTTGGTGAGGAGACATGGGAGAASCTGAAGGCTCOTGCAGAAGTGCAAGATGTCTTCATGACCTACACCGTGTATGATGACATCATCACCATTAAGCTCATCCAAGAAGCCTGCAAGGTTCTGGATGTGTCCATGGAAGCCATTCTGAAGCTCTTTGGCGAATACTTCTTTAAGTTCTGTAAGATGTCTGGCTATGACAGGATGCTGCGGACACTTGGAGGAAATCTCACCGAGTTTATTGAAAACCTAGATGCACTCCACAGTTACCTGGCACTGTCCTATCAGGAAATGAACGCACCATCCTTTCGAGTGGAGGAAGGAGCTGACGGGGCGATGCTTCTCCACTACTACTCAGACAGACATGGTCTGTGTCACATTGTACCAGGTATCATTGAAGCTGTGGCCAAGGACTTCTTTGACACTGATGTGGCCATGAGTATCCTGGATATGAACGAAGAGGTGGAAAGGACAGGGAAGAAAGAACATGTTGTGTTTCTGGTCGTGCAGAAGGCTCACAGACAGATAAGAGGAGCAAAGGCAAGCCGGCCACAAGGCAGTGAGGACAGCCAGGCAGACCAGGAGGCTCTCCAGGGAACACTCCTT(SEQ ID NO: 15)MYGFINTCLQSLVTEKFGEETWEKLKAPAEVQDVFMTYTVYDDIITIKLIQEACKVLDVSMEAILKLFGEYFFKFCKMSGYDRMLATLGGNLIEFIENLDALHSYLALSYQEMNAPSYAVEEGADGAMLLHYYSDRHGLCHIVPGIIEAVAKDFFDTDVAMSILDMNEEVERTGKKEHVVFLVVQKAHRQIRGAKASRPQGSEDSQADQEALQGILL(SEQ ID NO: 16) Rattus norvezieus β1 (1-385)ATGTACGGTTTTGTGAACCATGCCCTGGAGCTGCTGGTGATCCGCAATTACGGTCCCGAGGTGTGGGAAGACATCAAAAAAGAGGCGCAGCTGGATGAAGAAGGCCAGTTTCTTGTGAGAATAATCTACGATGATTCCAAAACCTATGACTTGGTGGCTGCTGCGAGCAAAGTCCTCAACCTCAATGCTGGTGAAATCCTGCAGATGTTTGGGAAGATGTTTTTCGTCTTCTGTCAAGAGTCTGGCTATGATACCATCTTGCGTGTCCTGGGATCTAATGTCAGGGAGTTTTTGCAGAACCTCGACGCCCTGCACGACCACCTCGCCACCATCTACCCAGGGATGCGCGCACCTTCCTTCCGGTGCACCGATGCAGAAAAAGGCAAAGGGCTCATTCTGCACTACTACTCGGAAAGAGAGGGGCTTCAGGACATTGTGATCGGGATTATCAAGACTGTAGCTCAACAGATCCATGGCACTGAGATAGACATGAAGGTTATTCAGCAAAGAAGTGAAGAATGTGATCATACCCAATTTTTAATTGAAGAAAAAGAATCAAAAGAAGAGGATTTTTATGAAGATCTGGACAGGTTTGAAGAGAACGGTACCCAGGACTCCCGTATCAGCCCGTACACCTTCTGCAAAGCGTTTCCTTTTCACATCATATTTGACCGGGACCTAGTAGTCACGCAGTGTGGAAATGCTATCTACAGAGTGCTCCCCCAGCTCCAGCCTGGGAAGTGCAGCCTTCTGTCTGTCTTCTCTCTGGTCCGCCCTCATATTGACATCAGTTTCCACGGGATTCTTTCACACATCAATACCGTCTTTGTACTGAGAAGCAAGGAAGGGTTGCTGGATGTTGAGAAACTTGAATGTGAGGATGAACTGACTGGGGCAGAGATTAGCTGCCTCCGTCTCAAAGGCCAAATGATCTATTTACCGGAAGGAGATAGCATCCTCTTCCTCTGTTCACCAAGTGTGATGAACTTGGATGACCTAACAAGAAGAGGCCTGTACCTGAGTGACATCCCTCTCCATGATGCTACACGAGACCTGGTCCTTTTGGGAGAACAGTTCCGGGAGGAGTACAAACTGACACAAGAGCTGGAAATCCICACAGACAGGCTGCAGCTCACACTGAGGGCTTTGGAGGATTGA (SEQ ID NO: 17)MYGFVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMFFVFCQESGYDTILRVLGSNVAEFLQNLDALHDHLATIYPGMRAPSFRCTDAEKGKGLILHYYSEREGLQDIVIGIIKTVAQQIHGTEIDMKVIQQRSEECDHTQFLIEEKESKEEDFYEDLDRFEENGTQCSRISPYTFCKAFPFHIIFDRDLVVTQCGNAIYRVLPQLQPGKCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDELTGAEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRAGLYLSDIPLHDATRDLVLLGEOFREEYKLTQELEILTDRLQLTLRALED (SEQ ID NO: 18) Rattus norvegicus β1 (1-385)ATGTACGGTTTTGTGAACCATGCCCTGGAGCTGCTGGTGATCCGCAATTACGGTCCCGAGGTGTGGGAAGACATCAAAAAAGAGGCGCAGCTGGATGAAGAAGGCCAGTTTCTTGTGAGAATAATCTACGATGATTCCAAAACCTATGACTTGGTGGCTGCTGCGAGCAAAGTCCTCAACCTCAATGCTGGTGAAATCCTGCAGATGTTTGGGAAGATGTTTTTCGTCTTCTGTCAAGAGTCTGGCTATGATACCATCTTGCGTGTCCTGGGATCTAATGTCAGGGAGTTTTTGCAGAACCTCGACGCCCTGCACGACCACCTCGCCACCATCTACCCAGGGATGCGCGCACCTTCCTTCCGGTGCACCGATGCAGAAAAAGGCAAAGGGCTCATTCTGCACTACTACTCGGAAAGAGAGGGGCTTCAGGACATTGTGATCGGGATTATCAAGACTGTAGCTCAACAGATCCATGGCACTGAGATAGACATGAAGGTTATTCAGCAAAGAAGTGAAGAATGTGATCATACCCAATTTTTAATTGAAGAAAAAGAATCAAAAGAAGAGGATTTTTATGAAGATCTGGACAGGTTTGAAGAGAACGGTACCCAGGACTCCCGTATCAGCCCGTACACCTTCTGCAAAGCGTTTCCTTTTCACATCATATTTGACCGGGACCTAGTAGTCACGCAGTGTGGAAATGCTATCTACAGAGTGCTCCCCCAGCTCCAGCCTGGGAAGTGCAGCCTTCTGTCTGTCTTCTCTCTGGTCCGCCCTCATATTGACATCAGTTTCCACGGGATTCTTTCACACATCAATACCGTCTTTGTACTGAGAAGCAAGGAAGGGTTGCTGGATGTTGAGAAACTTGAATGTGAGGATGAACTGACTGGGGCAGAGATTAGCTGCCTCCGTCTCAAAGGCCAAATGATCTATTTACCGGAAGCAGATAGCATCCTCTTCCTCTGTTCACCAAGTGTGATGAACTTGGATGACCTAACAAGAAGAGGCCTGTACCTGAGTGACATCCCTCTCCATGATGCTACACGAGACCTGGTCCTTTTGGGAGAACAGTTCCGGGAGGAGTACAAACTGACACAAGAGCTGGAAATCCTCACAGACAGGCTGCAGCTCACACTGAGGGCTTTGGAGGATTGA (SEQ ID NO: 19)MYGEVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMFFVFCQESGYDTILRVLGSNVREFLONLDALHDHLATIYPGMRAPSFRCTDAEKGKGLILHYYSEREGLQDIVIGIIKTVAQQIHGTEIDMKVIQQRSEECDHTQFLIEEKESKEEDFYEDLDRFEENGTQDSRISPYTFCKAFPFHIIFDRDLVVTQCGNAIYRVLPQLQPGKCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDELTGAEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRRGLYLSDIPLHDATRDLVLLGEQFREEYKLTQELEILTDRLQLTLRALED (SEQ ID NO: 20) Rattus norvegicus β2ATGTATGGATTCATCAACACCTGCCTGCAGTCTCTTGTGACAGAGAAATTTGGTGAGGAGACATGGGAGAAGCTGAAGGCTCCTGCAGAAGTGCAAGATGTCTTCATGACCTACACCGTGTATGATGACATCATCACCATTAAGCTCATCCAAGAAGCCTGCAAGGTTCTGGATGTGTCCATGGAAGCCATTCTGAAGCTCTTTGGCGAATACTTCTTTAAGTTCTGTAAGATGTCTGGCTATGACAGGATGCTGCGGACACTTGGAGGAAATCTCACCGAGTTTATTGAAAACCTAGATGCACTCCACAGTTACCTGGCACTGTCCTATCAGGAAATGAACGCACCATCCTTTCGAGTGGAGGAAGGAGCTGACGGGGCGATCCTTCTCCACTACTACTCAGACAGACATGGTCTGTGTCACATTCTACCAGGTATCATTGAAGCTGTGGCCAAGGACTTCTTTGACACTGATGTGGCCATGAGTATCCTGGATATGAACGAAGAGGTGGAAAGGACAGGGAAGAAAGAACATGTTGTGTTTCTGGTCGTGCAGAAGGCTCACAGACAGATAAGAGGAGCAAAGGCAAGCCGGCCACAAGGCAGTGAGGACAGCCAGGCAGACCAGGAGGCTCTCCAGGGAACACTCCTTCGGATGAAGGAGAGATATTTAAACATCCCTGTTTGCCCTGGGGAGAAATCTCACTCAACTGCTGTGAGGGCATCGGTCCTTTTTGGAAAAGGGCCCCTCAGGGACACCTTCCAGCCCGTCTATCCTGAGAGACTATGGGTCGAAGAGGAGGTGTTCTGTGATGCTTTTCCTTTCCACATTGTCTTTGATGAAGCACTAAGGGTCAAGCAAGCTGGAGTGAATATTCAGAAGTATGTCCCTGGAATCTTAACCCAGAAGTTTGCACTAGATGAGTATTTTTCCATCATCCACCCTCAAGTTACTTTCAACATCTCCAGCATCTGCAAGTTCATTAACAGTCAGTTTGTCTTGAAGACAAGAAAAGAAATGATGCCCAAAGCAAGGAAGAGCCAGCCGATGCTCAAACTCCGGGGTCAGATGATCTGGATGGAGTCTCTGAGGTGCATGATCTTCATGTGTTCCCCAAACGTCCGCAGCCTGCAAGAGCTGGAAGAGAGCAAGATGCATCTTTCTGATATCGCTCCGCACGACACGACCAGGGATCTCATCCTCCTCAACCAGCAGAGGCTGGCAGAGATGGAGCTGTCCTGCCAACTGGAAAAGAAGAAGGAGGAGTTGCGTGTCCTTTCCAATCACCTGGCCATCGAGAAGAAGAAGACAGAGACCTTGCTGTATGCCATGCTGCCTGAACATGTGGCCAACCAACTCAAGGAGGGCAGAAAGGTGGCTGCAGGAGAATTTGAAACATGTACAATCCTTTTCAGCGATGTTGTGACATTTACCAACATCTGTGCAGCCTGTGAACCTATCCAAATCGTGAACATGCTGAATTCAATGTACTCCAAGTTTGACAGGTTAACCAGTGTCCATGATGTCTACAAAGTAGAAACAATAGGGGATGCTTACATGGTGGTGGGTGGAGTACCAGTACCCGTTGAAAGCCATGCTCAAAGAGTCGCCAATTTTGCTCTGGGGATGAGAATTTCTGCAAAAGAAGTGATGAATCCTGTCACTGGGGAACCTATCCAGATCAGAGTGGGAATCCACACTGGACCAGTCTTAGCAGGTGTTGTGGGAGACAAGATGCCTCGGTACTGCTTGTTTGGTGACACTGTAAACACAGCCTCTAGGATGGAAAGTCACGGGCTTCCCAGCAAAGTGCATCTGAGCCCCACAGCCCACAGAGCCCTGAAAAACAAAGGGTTTGAAATTGTCAGGAGAGGCGAGATCGAAGTGAAGGGGAAAGGAAAGATGACCACATACTTTCTGATCCAGAACCTGAATGCCACCGAGGATGAGATAATGGGGCGACCTTCAGCCCCCGCTGATGGGAAGGAAGTATGTACTCCCGGAAACCAAGTCAGGAAGTCCCCTGCTGTCCCGAGGAACACAGACCATCAGCAACAAGTCTACAAAGGAGACCCAGCAGACGCTTCTAATGAAGTCACACTTGCTGGGAGCCCAGTGGCAGGGCGAAACTCCACAGATGCAGTCAATAACCAGCCATCACCAGATGAGACCAAGACAAGTGTCGTTGCTAGTGGCCCTGTGCTGTCTGCTTTCTGTGTTGTGCTGTGA (SEQ ID NO: 21)MYGFINTCLQSLVTEKFGEETWEKLKAPAEVQDVFMTYTVYDDIITIKLIQEACKVLDVSMEAILKLFGEYFFKFCKMSGYDRMLRTLGGNLTEFIENLDALHSYLALSYQEMNAPSFRVEEGADGAMLLHYYSDRHGLCHIVPGIIEAVAKDFFDTDVAMSILDMNEEVERTGKKEHVVFLVVQKAHRQIRGAKASRPQGSEDSQADQEALQGTLLRMKERYLNIPVCPGEKSHSTAVRASVLFGKGPLRDTFQPVYPERLWVEEEVFCDAFPFHIVFDEALRVKQAGVNIQKYVPGILTQKFALDEYFSIIHPQVTFNISSICKFINSQFVLKTRKEMMPKARKSQPMLKLRGQMIWMESLRCMIFMCLFEHVANOLKEGRKVAAGEFETCTILFSDVVIFTNICAACEPDOIVNMLNSMYSKFCRLTSVHDVYKVETIGDAYMVVGGVPVPVESHAQRVANFALGMRISAKEVMNPVTGEPIQRDVGIHTGPVLAGVVGDKMPRYCLFGDTVNTASRMESHGLPSKVHLSPTAHRALKNKGFEIVARGEIEVKGKGKMTTYFLIQNLNATECEIMGRPSAPADGKEVCTPGNOVRKSPAVPRNTDEQQQVYKGDPADASNEVTLAGSPVAGRNSTDAVNNQPSPDETKTSVVASGPVLSAFCVVL (SEQ ID NO: 22)

What is claimed is:
 1. A method for treating cancer in an individualcomprising administering to the individual an effective amount of an O₂carrier polypeptide.
 2. The method of claim 1, wherein the cancer isbrain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, melanoma, lung cancer, uterine cancer,ovarian cancer, colorectal cancer, anal cancer, liver cancer,hepatocellular carcinoma, stomach cancer, testicular cancer, endometrialcancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma,esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer,bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma,or squamous cell cancer.
 3. A method for modulating tumor immunity in anindividual with a tumor comprising administering to the individual aneffective amount of an O₂ carrier polypeptide.
 4. The method of claim 3,wherein the modulating tumor immunity comprises enhancing an immuneresponse to the tumor.
 5. A method for increasing lymphocyteinfiltration to a tumor in an individual comprising administering to theindividual an effective amount of an O₂ carrier polypeptide.
 6. Themethod of claim 5, wherein the increase in lymphocyte infiltration tothe tumor comprises an increase in infiltration of one or more of CD4cells, CD8 cells, or NK cells.
 7. The method of claim 5 or 6, whereinthe increase in lymphocyte infiltration to the tumor is accompanied byinhibition of one or more of Treg cells, tumor associated macrophages ormyeloid derived suppressor cells in the tumor.
 8. The method of any oneof claims 5-7, wherein the increase in lymphocyte infiltration to thetumor is accompanied by an increase in MHC1 expression on the tumorcells.
 9. A method for decreasing expression of hypoxia inducible factor1α (HIF-1α) and/or 2α (HIF-2α) in a tumor in an individual comprisingadministering to the individual an effective amount of an O₂ carrierpolypeptide.
 10. A method for decreasing expression of programmed deathligand-1 (PD-L1) in a tumor in an individual comprising administering tothe individual an effective amount of an O₂ carrier polypeptide.
 11. Amethod for decreasing expression of A2A adenosine receptor (A2AR) in atumor in an individual comprising administering to the individual aneffective amount of an O₂ carrier polypeptide.
 12. The method of any oneof claims 3-11, wherein the tumor is a brain tumor, a glioblastoma, abone tumor, a pancreatic tumor, a skin tumor, a tumor of the head orneck, a melanoma, a lung tumor, a uterine tumor, an ovarian tumor, acolorectal tumor, an anal tumor, a liver tumor, a hepatocellularcarcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, acervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin'slymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, anadrenal tumor, a bladder tumor, a kidney tumor, breast tumor, a multiplemyeloma tumor, a sarcoma, or a squamous cell tumor.
 13. The method ofany one of claims 1-12, wherein the individual is a mammal.
 14. Themethod of claim 13, wherein the mammal is a human.
 15. The method ofclaim 14, wherein the mammal is a pet, a laboratory research animal, ora farm animal.
 16. The method of claim 15, wherein the pet, researchanimal or farm animal is a dog, a cat, a horse, a monkey, a rabbit, arat, a mouse, a guinea pig, a hamster, a pig, or a cow.
 17. The methodof any one of claims 1-16, wherein the O₂ carrier polypeptide isadministered by intravenous, intra-arterial, intratumoral,intravesicular, inhalation, intraperitoneal, intrapulmonary,intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular,intrathecal, or transdermal administration.
 18. The method of any one ofclaims 1-17, wherein administration of the O₂ carrier polypeptide isrepeated.
 19. The method of claim 18, wherein administration of the O₂carrier polypeptide is repeated daily, twice a day or about 1-4 times aweek from about 4 weeks to about 8 weeks.
 20. The method of claim 18 or19 wherein the O₂ carrier polypeptide is administered every four, every8, every 12 or every 24 hours for a period of about one to about 10days.
 21. The method of any one of claims 1-20, wherein the O₂ carrierpolypeptide is administered as a bolus.
 22. The method of any one ofclaims 1-20, wherein the O₂ carrier polypeptide is administered byinfusion.
 23. The method of claim 22, wherein the 02 carrier polypeptideis infused in the individual for about 15 minutes, about 30 minutes,about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12hours or about 24 hours.
 24. The method of any one of claims 1-23,wherein the O₂ carrier polypeptide is administered in combination withradiation therapy.
 25. The method of claim 24, wherein the radiationtherapy is administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12,14, 16, 18, 20 or 24 hours after the O₂ carrier polypeptide isadministered.
 26. The method of claim 24 or 25, wherein the radiation isX-radiation.
 27. The method of claim 26, wherein the X-radiation isadministered at about 0.5 gray to about 75 gray.
 28. The method of anyone of claims 24-27, wherein the administration of the O₂ carrierpolypeptide and/or the administration of the radiation is repeated. 29.The method of claim 28, wherein the administration is repeated anynumber of times between about two times to about forty times or more.30. The method of claim 28 or 29, wherein the administration is repeatedafter one week, two weeks, three weeks, or four weeks or more.
 31. Themethod of any one of claims 1-23, wherein the O₂ carrier polypeptide isadministered in combination with chemotherapy or immunotherapy.
 32. Themethod of claim 31, wherein the chemotherapy comprises a cytotoxin. 33.The method of claim 32, wherein the administration of the O₂ carrierpolypeptide and/or the administration of the chemotherapy is repeated.34. The method of claim 31, wherein the immunotherapy is one or more ofan anticancer vaccine, an adoptive immune cell therapy or an agent thattargets an immune checkpoint regulator.
 35. The method of claim 31 or34, wherein the immunotherapy targets one or more of CTLA-4, PD1, PD-L1,or an immune checkpoint regulator.
 36. The method of claim 31 or 34,wherein the adoptive immude therapy is a chimeric antigen receptorexpressing T cell or an engineered TCR-T cell.
 37. The method of any oneof claims 31 or 34-36, wherein the administration of the O₂ carrierpolypeptide and/or the administration of the immunotherapy is repeated.38. The method of any one of claims 1-37, wherein the O₂ carrierpolypeptide is in a pharmaceutical composition.
 39. The method of claim38, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 40. The method of any one of claims1-39 wherein the O₂ carrier polypeptide is an H-NOX protein.
 41. Amethod for treating cancer in an individual comprising administering tothe individual an effective amount of an H-NOX protein.
 42. The methodof claim 41, wherein the cancer is brain cancer, glioblastoma, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,melanoma, lung cancer, uterine cancer, ovarian cancer, colorectalcancer, anal cancer, liver cancer, hepatocellular carcinoma, stomachcancer, testicular cancer, endometrial cancer, cervical cancer,Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal cancer, intestinalcancer, thyroid cancer, adrenal cancer, bladder cancer, kidney cancer,breast cancer, multiple myeloma, sarcoma, or squamous cell cancer.
 43. Amethod for modulating tumor immunity in an individual with a tumorcomprising administering to the individual an effective amount of anH-NOX protein.
 44. The method of claim 43, wherein the modulating tumorimmunity comprises enhancing an immune response to the tumor.
 45. Amethod for increasing lymphocyte infiltration to a tumor in anindividual comprising administering to the individual an effectiveamount of an H-NOX protein.
 46. The method of claim 45, wherein theincrease in lymphocyte infiltration to the tumor comprises an increasein infiltration of one or more of CD4 cells, CD8 cells, or NK cells. 47.The method of claim 45 or 46, wherein the increase in lymphocyteinfiltration to the tumor is accompanied by inhibition of one or more ofTreg cells, tumor associated macrophages or myeloid derived suppressorcells in the tumor.
 48. The method of any one of claims 45-47, whereinthe increase in lymphocyte infiltration to the tumor is accompanied byan increase in MHC1 expression on the tumor cells.
 49. A method fordecreasing expression of HIF-1α and/or HIF-2α in a tumor in anindividual comprising administering to the individual an effectiveamount of an H-NOX protein.
 50. A method for decreasing expression ofPD-L1 in a tumor in an individual comprising administering to theindividual an effective amount of an H-NOX protein.
 51. A method fordecreasing expression of A2AR in a tumor in an individual comprisingadministering to the individual an effective amount of an H-NOX protein.52. The method of any one of claims 41-51, wherein the tumor is a braintumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, atumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, anovarian tumor, a colorectal tumor, an anal tumor, a liver tumor, ahepatocellular carcinoma, a stomach tumor, a testicular tumor, anendometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin'slymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinaltumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidneytumor, a breast tumor, a multiple myeloma tumor, a sarcoma, or asquamous cell tumor.
 53. The method of any one of claims 41-52, whereinthe individual is a mammal.
 54. The method of claim 53, wherein themammal is a human.
 55. The method of claim 52, wherein the mammal is apet, a laboratory research animal, or a farm animal.
 56. The method ofclaim 55, wherein the pet, research animal or farm animal is a dog, acat, a horse, a monkey, a rabbit, a rat, a mouse, a guinea pig, ahamster, a pig, or a cow.
 57. The method of any one of claims 41-56,wherein the H-NOX protein is administered by intravenous,intra-arterial, intratumoral, intravesicular, inhalation,intraperitoneal, intrapulmonary, intramuscular, subcutaneous,intra-tracheal, transmucosal, intraocular, intrathecal, or transdermaladministration.
 58. The method of any one of claims 41-57, whereinadministration of the H-NOX protein is repeated.
 59. The method of claim58, wherein administration of the H-NOX protein is repeated daily ortwice a day from about 4 weeks to about 8 weeks.
 60. The method of claim58 or 59 wherein the H-NOX protein is administered every four, every 8,every 12 or every 24 hours for a period of about one to about 10 days.61. The method of any one of claims 41-60, wherein the H-NOX protein isadministered as a bolus.
 62. The method of any one of claims 41-60,wherein the H-NOX protein is administered by infusion.
 63. The method ofclaim 62, wherein the H-NOX protein is infused in the individual forabout 15 minutes, about 30 minutes, about 1 hour, about 1 hour, about 2hours, about 3 hours, about 6 hours, about 12 hours or about 24 hours.64. The method of any one of claims 41-63, wherein the H-NOX protein isadministered in combination with radiation therapy.
 65. The method ofclaim 64, wherein the radiation therapy is administered to theindividual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hoursafter the H-NOX protein is administered.
 66. The method of claim 64 or65, wherein the radiation is X-radiation.
 67. The method of claim 66,wherein the X-radiation is administered at about 0.5 gray to about 75gray.
 68. The method of any one of claims 64-67, wherein theadministration of the H-NOX protein and/or the administration of theradiation is repeated.
 69. The method of claim 68, wherein theadministration is repeated any number of times between about two timesto about forty times or more.
 70. The method of claim 68 or 69, whereinthe administration is repeated after one week, two weeks, three weeks,or four weeks or more.
 71. The method of any one of claims 41-63,wherein the H-NOX protein is administered in combination withchemotherapy or immunotherapy.
 72. The method of claim 71, wherein thechemotherapy comprises a cytotoxin.
 73. The method of claim 72, whereinthe administration of the H-NOX protein and/or the administration of thechemotherapy is repeated.
 74. The method of claim 71, wherein theimmunotherapy is one or more of an anticancer vaccine, an adoptiveimmune cell therapy or an agent that targets an immune checkpointregulator.
 75. The method of claim 71 or 74, wherein the immunotherapytargets one or more of CTLA-4, PD1, PD-L1, or an immune checkpointregulator.
 76. The method of claim 71, 74 or 75, wherein the adoptiveimmude therapy is a chimeric antigen receptor expressing T cell or anengineered TCR-T cell.
 77. The method of any one of claims 71, or 74-76,wherein the administration of the H-NOX protein and/or theadministration of the immunotherapy is repeated.
 78. The method of anyone of claims 41-77, wherein the H-NOX protein is a T. tengcongensisH-NOX, a L. pneumophilia 2 H-NOX, a H. sapiens β1, a R. norvegicus β1, aC. lupus H-NOX, a D. melangaster β1, a D. melangaster CG14885-PA, a C.elegans GCY-35, a N. punctiforme H-NOX, C. crescentus H-NOX, a S.oneidensis H-NOX, or C. acetobutylicum H-NOX.
 79. The method of any oneof claims 41-77, wherein the H-NOX protein comprises a H-NOX domaincorresponding to the H-NOX domain of T. tengcongensis set forth in SEQID NO:2.
 80. The method of any one of claims 41-78, wherein the H-NOXcomprises one or more distal pocket mutations.
 81. The method of claim80, wherein the distal pocket mutation is an amino acid substitution ata site corresponding to L144 of T. tengcongensis H-NOX.
 82. The methodof claim 80 or 81, wherein the H-NOX is a T. tengcongensis H-NOXcomprising an amino acid substitution at position
 144. 83. The method ofclaim 82, wherein the amino acid substitution at position 144 is anL144F substitution.
 84. The method of any one of claims 41-83, whereinthe H-NOX protein is a polymeric H-NOX protein.
 85. The method of claim84, wherein the polymeric H-NOX protein comprises monomers, wherein themonomers comprise an H-NOX domain and a polymerization domain.
 86. Themethod of claim 85, wherein the H-NOX domain is covalently linked to thepolymerization domain.
 87. The method of any one of claims 84-86,wherein the polymeric H-NOX protein is a trimeric H-NOX protein.
 88. Themethod of claim 87, wherein the trimeric H-NOX protein comprises one ormore trimerization domains.
 89. The method of claim 88, wherein thetrimeric H-NOX protein comprises three monomers, wherein the monomerscomprise an H-NOX domain and a trimerization domain, wherein thetrimerization domain is a bacteriophage T4 trimerization domain.
 90. Themethod of claim 88 or 89, wherein the trimerization domain is a foldondomain.
 91. The method of claim 90, wherein the foldon domain comprisesthe amino acid sequence of SEQ ID NO:4.
 92. The method of any one ofclaims 41-91, wherein the H-NOX protein is fused to an Fc domain of animmunoglobulin.
 93. The method of any one of claims 41-92, wherein theH-NOX protein is covalently bound to polyethylene glycol.
 94. The methodof any one of claims 41-93, wherein the O₂ dissociation constant of theH-NOX protein is within 2 orders of magnitude of that of hemoglobin, andwherein the NO reactivity of the H-NOX protein is at least 10-fold lowerthan that of hemoglobin.
 95. The method of any one of claims 41-94,wherein the O₂ dissociation constant of the polymeric H-NOX protein isbetween about 1 nM and about 1000 nM at 20° C.
 96. The method of any oneof claims 41-95, wherein the O₂ dissociation constant of the H-NOXprotein is between about 1 μM and about 10 μM at 20° C.
 97. The methodof any one of claims 41-96, wherein the Oz dissociation constant of theH-NOX protein is between about 10 μM and about 50 μM at 20° C.
 98. Themethod of any one of claims 41-97, wherein the NO reactivity of theH-NOX protein is less than about 700 s⁻¹ at 20° C.
 99. The method of anyone of claims 41-98, wherein the NO reactivity of the H-NOX protein isat least 100-fold lower than that of hemoglobin.
 100. The method ofclaim 99, wherein the NO reactivity of the H-NOX protein is at least1,000-fold lower than that of hemoglobin.
 101. The method of any one ofclaims 41-100, wherein the k_(off) for oxygen of the H-NOX protein isless than or equal to about 0.65 s⁻¹ at 20° C.
 102. The method of anyone of claims 41-101, wherein the k_(off) for oxygen of the H-NOXprotein is between about 0.21 s⁻¹ and about 0.65 s⁻¹ at 20° C.
 103. Themethod of any one of claims 41-102, wherein the k_(off) for oxygen ofthe H-NOX protein is between about 1.35 s⁻¹ and about 2.9 s⁻¹ at 20° C.104. The method of any one of claims 41-103, wherein the rate of hemeautoxidation of the H-NOX protein is less than about 1 h⁻¹ at 37° C.105. The method of any one of claims 41-104, wherein the H-NOX proteinis in a pharmaceutical composition.
 106. The method of claim 105,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 107. Use of an O₂ carrier proteinfor treating cancer in an individual.
 108. The use of claim 107, whereinthe cancer is brain cancer, glioblastoma, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, melanoma, lung cancer,uterine cancer, ovarian cancer, colorectal cancer, anal cancer, livercancer, hepatocellular carcinoma, stomach cancer, testicular cancer,endometrial cancer, cervical cancer, Hodgkin's Disease, non-Hodgkin'slymphoma, esophageal cancer, intestinal cancer, thyroid cancer, adrenalcancer, bladder cancer, kidney cancer, breast cancer, multiple myeloma,sarcoma, or squamous cell cancer.
 109. Use of an O₂ carrier protein formodulating tumor immunity in an individual.
 110. The use of claim 109,wherein the modulating tumor immunity comprises enhancing an immuneresponse to the tumor.
 111. Use of an O₂ carrier polypeptide forincreasing lymphocyte infiltration to a tumor in an individual.
 112. Theuse of claim 111, wherein the increase in lymphocyte infiltration to thetumor comprises an increase in infiltration of one or more of CD4 cells,CD8 cells, or NK cells.
 113. The use of claim 111 or 112, wherein theincrease in lymphocyte infiltration to the tumor is accompanied byinhibition of one or more of Treg cells, tumor associated macrophages ormyeloid derived suppressor cells in the tumor.
 114. The use of any oneof claims 111-113, wherein the increase in lymphocyte infiltration tothe tumor is accompanied by an increase in MHC1 expression on the tumorcells.
 115. Use of an O₂ carrier polypeptide for decreasing expressionof HIF-1α and/or HIF-2α in a tumor in an individual.
 116. Use of an O₂carrier polypeptide for decreasing expression of PD-L1 in a tumor in anindividual.
 117. Use of an O₂ carrier polypeptide for decreasingexpression of A2AR in a tumor in an individual.
 118. The use of any oneof claims 109-117, wherein the tumor is a brain tumor, a glioblastoma, abone tumor, a pancreatic tumor, a skin tumor, a tumor of the head orneck, a melanoma, a lung tumor, a uterine tumor, an ovarian tumor, acolorectal tumor, an anal tumor, a liver tumor, a hepatocellularcarcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, acervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin'slymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, anadrenal tumor, a bladder tumor, a kidney tumor, a breast tumor, amultiple myeloma tumor, a sarcoma, or a squamous cell tumor.
 119. Theuse of any one of claims 107-118, wherein the individual is a mammal.120. The use of claim 119, wherein the mammal is a human.
 121. The useof any one of claims 107-120, wherein the O₂ carrier polypeptide is anH-NOX protein.
 122. The use of claim 121, wherein the H-NOX protein is aT. tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a H. sapiens β1, a R.norvegicus β1, a C. lupus H-NOX domain, a D. melangaster β1, a D.melangaster CG14885-PA, a C. elegans GCY-35, a N. punctiforme H-NOX, C.crescentus H-NOX, a S. oneidensis H-NOX, or C. acetobutylicum H-NOX.123. The use of any one of claims 121-122, wherein the H-NOX proteincomprises a H-NOX domain corresponding to the H-NOX domain of T.tengcongensis set forth in SEQ ID NO:2.
 124. The use of any one ofclaims 121-123, wherein the H-NOX comprises one or more distal pocketmutations.
 125. The use of claim 124, wherein the distal pocket mutationis an amino acid substitution at a site corresponding to L144 of T.tengcongensis H-NOX.
 126. The use of claim 124 or 125, wherein the H-NOXis a T. tengcongensis H-NOX comprising an amino acid substitution atposition
 144. 127. The use of claim 126, wherein the amino acidsubstitution at position 144 is an L144F substitution.
 128. The use ofany one of claims 121-127, wherein the H-NOX protein is a polymericH-NOX protein.
 129. The use of claim 128, wherein the polymeric H-NOXprotein comprises monomers, wherein the monomers comprise an H-NOXdomain and a polymerization domain.
 130. The use of claim 129, whereinthe H-NOX domain is covalently linked to the polymerization domain. 131.The use of any one of claims 128-130, wherein the polymeric H-NOXprotein is a trimeric H-NOX protein.
 132. The use of claim 131, whereinthe trimeric H-NOX protein comprises one or more trimerization domains.133. The use of claim 132, wherein the trimeric H-NOX protein comprisesthree monomers, wherein the monomers comprise an H-NOX domain and atrimerization domain, wherein the trimerization domain is abacteriophage T4 trimerization domain.
 134. The use of claim 132 or 133,wherein the trimerization domain is a foldon domain.
 135. The use ofclaim 134, wherein the foldon domain comprises the amino acid sequenceof SEQ ID NO:4.
 136. The use of any one of claims 131-135, wherein theH-NOX protein is fused to an Fc domain of an immnunoglobulin.
 137. Theuse of any one of claims 121-136, wherein the H-NOX protein iscovalently bound to polyethylene glycol.
 138. A kit for modulating tumorimmunity in an individual comprising an O₂ carrier protein for use inthe method of any one of claims 1-106.
 139. The kit of claim 138,wherein the kit further comprises one or more of a vial, a vessel, anampule, a bottle, a jars, or flexible packaging.
 140. The kit of claim138 or 139, wherein the kit further comprises one or more buffers. 141.The kit of any one of claims 138-140, wherein the kit further comprisesinstructions for use.